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i
Anna Lundin
Department of Infectious Diseases
Institute of Biomedicine
Sahlgrenska Academy at University of Gothenburg
Gothenburg 2013
ii
Cover illustration: Perinuclear clusters of the double membrane vesicles
induced by human coronavirus 229E in lung fibroblasts. Prepared by Anna
Lundin and Sibylle Widehn
Candidate antivirals for treatment of respiratory syncytial virus and
coronavirus infections
© Anna Lundin 2013
ISBN 978-91-628-8780-3
ISBN 978-91-628-8782-7 (pdf)
http://hdl.handle.net/2077/33097
Printed in Gothenburg, Sweden 2013
Printed by Kompendiet
iii
iv
v
Anna Lundin
Department of Infectious Diseases, Institute of Biomedicine
Sahlgrenska Academy at University of Gothenburg
Göteborg, Sweden
ABSTRACT
Respiratory syncytial virus (RSV) and coronaviruses (CoVs) are frequent
causes of respiratory disease in humans. RSV can cause severe bronchiolitis
and pneumonia in infants, especially in those born prematurely or with
underlying cardiopulmonary chronic dysfunction. CoV respiratory illnesses
can vary in severity ranging from common cold-like symptoms to severe
respiratory disease with potential fatal outcome as exemplified by the
pandemic-causing SARS- or MERS-CoVs. Despite the frequency and
severity of RSV and CoV diseases, attempts to develop an effective and
non-toxic antiviral treatment or a vaccine have so far been unsuccessful.
The aim of this thesis was to identify new antiviral candidates for treatment
of RSV and CoV infections, and to elucidate their antiviral mechanism.
Through screening of the ChemBioNet collection of ~17000 diverse
compounds and a mini-library of polysulfated tetra- and pentasaccharide
glycosides in a cell culture-based whole virus system, three promising anti-
RSV and one anti-CoV candidate drugs were identified. Subsequent
application of our step-by-step assay strategy for elucidation of mode-of-
antiviral activity (paper III), revealed that anti-RSV P13 and C15
compounds displayed potent antiviral activity by targeting the heptad repeat
regions of the viral F-protein essential for the virus-cell and the cell-cell
membrane fusion (paper I). The anti-RSV lead drug PG545, identified in
paper II, was prepared by coupling of a lipophilic cholestanol group to the
synthetic sulfated oligosaccharide. This modification of the oligosaccharide
enhanced the anti-RSV activity and conferred virucidal properties on
PG545, a feature absent in native sulfated oligosaccharide inhibitors (paper
II). PG545 exhibited dual antiviral mechanisms including (i) reduction of
the RSV attachment to cells due to targeting of the highly conserved region
and the receptor-binding region of the viral attachment G-protein, and (ii)
vi
direct inactivation of viral particles. The anti-CoV candidate drug K22
potently inhibited 229E-CoV infectivity by targeting the membrane-bound
viral RNA synthesis in the cytoplasm (paper IV). Analysis of viral variants
resistant to K22 in addition to the preparation of specific 229E-recombinant
viruses, revealed that K22 targets the viral nonstructural protein 6 (nsp6).
This protein is involved in the recruitment and modification of host cellular
membranes to create sites for the virus membrane-bound RNA synthesis.
This is the first report of nsp6 as a druggable target for CoV intervention.
K22 was also shown to be active against many other CoVs including the
newly emerged MERS-CoV. In conclusion, P13, C15, PG545, and K22 are
promising candidates for further development as new anti-RSV and anti-
CoV drugs.
Keywords: antivirals, respiratory syncytial virus, coronavirus, antiviral
screening, fusion inhibitors, sulfated oligosaccharides, nsp6, membrane
vesicles
ISBN: 978-91-628-8780-3
vii
Respiratoriskt syncytialt virus (RSV) och coronavirus (CoV) utgör två av
våra vanligaste orsaker till luftvägsinfektion hos människa. RSV kan orsaka
lunginflammation hos spädbarn och då speciellt hos barn som är för tidigt
födda eller har en underliggande kronisk hjärt- eller lungsjukdom. Även äldre
drabbas i hög utsträckning av RSV, också här ofta i form av
lunginflammation. Luftvägsinfektioner orsakade av CoV kan vara mer eller
mindre allvarliga och orsaka allt från vanliga förkylningssymptom som
kroppen själv läker ut till mer omfattande infektioner som i de svåraste fallen
kräver sjukhusvård och ibland även leder till dödsfall. Exempel på CoV som
kan resultera i sådana kraftigare luftvägsinfektioner är det pandemiska
SARS-CoV, samt det nyligen identifierade MERS-CoV.
Det finns i nuläget ingen godkänd antiviral behandling mot infektioner
orsakade av CoV och endast ett läkemedel finns tillgängligt för behandling av
RSV-infektion. Detta läkemedel är relativt dyrt och ger dessvärre inte
tillräcklig klinisk effekt. Trots att båda dessa virusgrupper frekvent orsakar
allvarliga luftvägsinfektioner har försöken att för att ta fram nya antivirala
behandlingsmöjligheter hittills inte resulterat i något nytt läkemedel.
Syftet med den här avhandlingen var att hitta nya kandidat-substanser för
behandling av infektioner orsakade av RSV och CoV. Ett cellkultur-baserat
metodologiskt flerstegssystem upprättades för att systematiskt undersöka de
mest lovande kandidaterna samt för att ta reda på hur de hämmar
virusinfektionen och vilken del av virus eller dess infektionscykel som
blockeras, dvs. för att definiera deras antivirala mekanism. Genom att utgå
ifrån molekylära bibliotek testades, ”screenades”, en stor mängd substanser,
dels från ChemBioNet biblioteket på ca 17000 kemiska strukturer och även
ett mini-bibliotek bestående av högsulfaterade kolhydrater. Studierna
genomfördes inledningsvis i en modell där levande celler med ursprung från
luftvägarna infekterades med virus i närvaro av en substans för att se om
denna kunde skydda cellerna mot infektion. Den etablerade strategin för att
utvärdera virushämmande mekanism beskrivs utförligt i arbete III. Projektet
ledde till att vi här rapporterar om fyra nya lovande substanser, tre riktade
mot RSV och en mot CoV, som effektivt hämmade dessa virusinfektioner in
vitro.
I det första arbetet (I) identifierade vi substanserna P13 och C15 som
hämmade RSV-infektion genom att blockera funktionen av höljeproteinet F,
som virus använder för att ta sig in i värdcellen, och även för att sprida sig
viii
mellan celler. Ytterligare en RSV-hämmande substans, PG545, identifierades
i arbete II bland en grupp högsulfaterade kolhydrater som modifierats med
olika lipofila grupper. När en specifik tetrasackarid kopplades till en lipofil
kolestanolgrupp påvisades en starkt förbättrad blockering av RSV-infektion
jämfört med den okonjugerade ursprungssubstansen. PG545 påverkade
virusets förmåga att binda till celler genom att störa funktionen hos RSVs
höljeprotein G som förmedlar cellbindning, men även genom att interagera
med andra virusspecifika strukturer. Den lipofila gruppen medförde även att
substansen kunde inaktivera viruspartiklarna, en egenskap som helt saknades
hos de okonjugerade sulfaterade kolhydraterna. Anti-CoV-substansen K22
identifierades i arbete IV och hämmade effektivt infektion av CoV-stammen
229E genom att störa virus membranbundna RNA-syntes i cellens
cytoplasma. Virusvarianter resistenta mot K22 uppvisade genförändringar i
det icke-strukturella proteinet nsp6 vilket indikerade att denna
viruskomponent var målet för den virushämmande effekten. Med revers
genetik konstruerades tre rekombinanta CoV-229E, där resistensmutationerna
introducerades, och funktionella studier av dessa virus bekräftade att nsp6
utgjorde det antivirala målet. Virusproteinet deltar i skapandet av ”fabriker”
för CoV membranbundna replikation. Fyndet, som är det första i sitt slag
beskriver nsp6 som ett nytt mål för antiviral intervention riktad mot
infektioner orsakade av CoV. Vidare hämmade K22 cellulär infektion av
flera andra CoVs, däribland SARS-CoV och det nyligen upptäckta MERS-
viruset. Sammantaget utgör P13, C15, PG545 och K22 lovande kandidater
för vidare utveckling av nya antiviraler riktade mot CoV- och RSV-orsakade
infektioner.
ix
This thesis is based on the following studies, referred to in the text by their
Roman numerals.
I. Lundin A, Bergström T, Bendrioua L, Kann N, Adamiak B,
Trybala E. Two novel fusion inhibitors of human respiratory
syncytial virus. Antiviral Res 2010; 88(3):317-24.
II. Lundin A, Bergström T, Andrighetti-Fröhner CR, Bendrioua L,
Ferro V, Trybala E. Potent anti-respiratory syncytial virus activity
of a cholestanol-sulfated tetrasaccharide conjugate. Antiviral Res
2012; 93(1):101-9.
III. Lundin A, Bergström T, Trybala E. Screening and evaluation of
anti-respiratory syncytial virus compounds in cultured cells.
Methods Mol Biol 2013; 1030:345-63.
IV. Lundin A, Dijkman R, Bergström T, Kann N, Adamiak B,
Hannoun C, Kindler E, Jónsdóttir HR, Muth D, Kint J, Forlenza M,
Müller MA, Drosten C, Thiel V, Trybala E. Targeting membrane-
bound viral RNA synthesis reveals potent inhibition of diverse
coronaviruses, including the Middle East respiratory syndrome
virus. Submitted.
x
ABBREVIATIONS ........................................................................................... XIII
1 INTRODUCTION ........................................................................................... 1
1.1 Virus respiratory infections ................................................................... 1
1.1.1 RSV disease ................................................................................... 3
1.1.2 CoV disease ................................................................................... 4
1.2 Respiratory syncytial virus (RSV) ........................................................ 6
1.2.1 RSV particle .................................................................................. 6
1.2.2 RSV attachment to and fusion of cells - The G- and F-proteins ... 7
1.2.3 RSV replication ........................................................................... 12
1.3 Human respiratory coronaviruses (CoVs) ........................................... 14
1.3.1 The CoV particle ......................................................................... 14
1.3.2 Components of the CoV particle and their activities in viral life
cycle ............................................................................................ 15
1.3.3 Membrane-bound CoV RNA synthesis ....................................... 16
1.4 Respiratory epithelium, a target of RSV and CoV infections ............. 20
1.4.1 Structure and basic function of the human respiratory
epithelium .................................................................................... 20
1.4.2 Respiratory secretions – Physiological function and pathogen
defense ......................................................................................... 20
1.4.3 Epithelial cell surface receptors and CoV/RSV tropism ............. 23
1.5 Antiviral strategies .............................................................................. 26
1.5.1 Adverse effect of putative antiviral compounds on cells ............ 27
1.5.2 Virus life cycle events as targets for antiviral intervention ......... 27
xi
1.5.3 Antiviral drug resistance .............................................................. 31
1.6 Searching for anti-RSV and anti-CoV intervention ............................ 32
1.6.1 RSV vaccine and prophylaxis ..................................................... 32
1.6.2 RSV antivirals ............................................................................. 33
1.6.3 CoV vaccine and prophylaxis ...................................................... 36
1.6.4 CoV antivirals .............................................................................. 36
1.7 High throughput screening .................................................................. 39
2 AIMS ......................................................................................................... 41
3 MATERIALS AND METHODS ..................................................................... 42
3.1 Collections (libraries) of compounds for antiviral screening .............. 42
3.2 Viruses and cells ................................................................................. 42
3.3 Antiviral screening and initial assessment of antiviral activity and
cytotoxicity of hits ...................................................................................... 43
3.4 Cell toxicity and proliferation assays .................................................. 45
3.5 Elucidation of compound mode-of-action ........................................... 45
3.5.1 Time-of- addition/removal, and virucidal assays ........................ 46
3.5.2 Attachment and fusion inhibition assays ..................................... 48
3.5.3 Classical time-of-addition assay/impact on viral RNA
synthesis ...................................................................................... 49
3.5.4 Generation of drug resistant viral variants .................................. 49
3.5.5 Generation of recombinant viral variants and their replication
fitness ........................................................................................... 50
3.5.6 Electron microscopy .................................................................... 50
4 RESULTS AND DISCUSSION ....................................................................... 52
4.1 Paper I and III...................................................................................... 52
xii
4.2 Paper II and III .................................................................................... 56
4.3 Paper IV .............................................................................................. 61
5 CONCLUDING REMARKS AND FUTURE PERSPECTIVES .............................. 69
ACKNOWLEDGEMENTS .................................................................................. 71
REFERENCES .................................................................................................. 74
xiii
aa Amino acids
CM Convoluted membranes
CPE Cytopathic effect
DMV Double membrane vesicle
DNA Deoxyribonucleic acid
dsRNA Double stranded ribonucleic acid
FCoV Feline Coronavirus
EM Electron microscopy
ER Endoplasmic reticulum
GAG Glycosaminoglycan
HAE Human airway epithelium
HBD Heparin binding domain
HCoV Human coronavirus
hMPV Human metapneumovirus
HR Heptad repeat
HS Heparan sulfate
HTS High throughput screening
IBV Infectious bronchitis virus
IC50 50% inhibitory concentration
IF Immunofluorescence
KS Keratan sulfate
MERS Middle East respiratory syndrome
MHV Mouse hepatitis virus
xiv
mRNA Messenger ribonucleic acid
Nsp6 Nonstructural protein 6
PFU Plaque forming unit
RC Replication complex
RdRp RNA-dependent RNA polymerase
RSV Respiratory syncytial virus
RTC Replication/transcription complex
RT-PCR Reverse transcriptase polymerase chain reaction
SARS Severe acute respiratory syndrome
TM Transmembrane
VP Vesicle packets
Anna Lundin
1
Acute respiratory infections have a significant health impact on individuals of
all ages and represents one of the major disease burdens worldwide which led
to 3.2 million fatalities in 2011 [1, 2]. Furthermore, acute respiratory
infections are frequent causes of mortality in children under the age of five
accounting for approximately 20% of all deaths [3]. WHO predicts that lower
respiratory tract infections will still be among the top five leading causes of
child death in the period of 2010 -2030 [4, 5]. A substantial portion of the
reported cases of severe respiratory tract infections are caused by viruses [6-
8]. There are a wide range of different respiratory viruses that constantly
circulate in the population causing respiratory illness of varying severity. The
common cold is a collection term for a similar set of symptoms including
sneezing, nasal obstruction, sore throat and coughing, caused by a range of
viruses belonging to several different families. In humans, common colds are
frequently caused by RNA viruses with rhinoviruses being among the major
causative agents [9-11]. In addition, viruses such as respiratory syncytial
virus (RSV), human metapneumovirus (hMPV), influenza virus,
parainfluenza viruses, and some adeno-, corona-, and enteroviruses, can to a
varying degree be associated with common cold symptoms.
In spite of the generally mild symptoms and self-clearing illness caused by
respiratory viruses, these pathogens can spread into the lower respiratory tract
and other organs causing severe, frequently fatal disease. Due to the
preferential tropism of these pathogens for respiratory epithelium, the
physical barriers of the respiratory tract and competent innate and adaptive
immunity are crucial for preventing the infection or restricting its progression
into severe respiratory disease. An example of this is infants and children
born prematurely, who due to narrow and not fully developed airways are
vulnerable to their obstruction following infection and inflammation. In
addition, their immature innate and adaptive immunity, especially that
mediated by the T helper 1 (Th1) cells, lacks the strength and capacity to
withhold respiratory pathogens and the infections tend to be prolonged and
more severe compared to those occurring in older children [12-14].
RSV is considered the sole most important viral pathogen causing lower
respiratory tract infections in infants and one of the major causes of mortality
in children under the age of five [7, 15]. It was estimated that in the year
Candidate antivirals for treatment of respiratory syncytial virus and coronavirus infections
2
2005 RSV accounted for 33.8 million, 22%, of all acute lower respiratory
tract infections in children less than five years of age worldwide [7]. A local
study of the incidence of RSV in Sweden coincides with the observation that
the age group of 0-4 years has the highest prevalence of RSV infections
accounting for almost 20% of all the positive cases in the children studied
[11]. There are two subgroups of RSV, A and B, that cause respiratory
infection in humans. Several studies indicate that infection with subtype A
results in a more severe disease outcome [16, 17]. Members of both
subgroups circulate annually and can appear simultaneously or most often
with dominance of one subtype [18, 19].
Human coronaviruses (CoVs) cause approximately 5 to 30% of all upper
respiratory infections in adults [9, 20-24]. There are six species of the
Coronaviridae family that are known to infect humans, and the two most
common, 229E and OC43, causing generally mild respiratory symptoms,
were identified in 1966 and 1967 respectively [25, 26]. In 2003 a new
member of the CoV family, SARS-CoV, caused a pandemic outbreak of
severe lower respiratory tract infection affecting more than 8000 individuals
with subsequent 750 fatalities [27-30]. CoV NL-63 and HKU1 were
identified as late as 2004 and 2005 in the aftermath of the SARS-CoV
outbreak which led to an immense increase in the efforts to characterize this
new pandemic virus [31, 32]. In addition to the SARS-CoV, a new potential
pandemic-causing MERS-CoV was identified 2012 in a 60-year old man
from Saudi-Arabia. The patient suffered from acute pneumonia and renal
failure and died 18 days after the onset of symptoms. Although the MERS-
and SARS-CoV share some similarities as regards zoonotic origin and
pathogenicity of the disease, the former virus is clearly distinct from the other
human CoVs [33]. Recently, a report of a hospital outbreak of MERS-CoV
has confirmed the person-to-person spread of this virus [34, 35]. At present,
there are 114 confirmed cases of MERS-CoV disease including 54 fatalities
confirming that this virus can be extremely deadly for humans (CDC, MERS-
CoV update 17th of September 2013) [36].
There is a clear seasonality to most respiratory virus infections in northern
temperate regions with a five to six month period ranging from late autumn
to early spring with peaks occurring at different times around the winter
months depending on the virus. A study of the seasonal variations of 15
different respiratory pathogens over a three year period in Sweden showed
that human rhinovirus was the most prevalent irrespective of the season,
followed by influenza A and RSV, both most prevailing from January to
March [11]. Every other year RSV tends to have a shorter peak season and
the cases seem to be less severe [37].
Anna Lundin
3
The human CoVs 229E, OC43, NL-63 and HKU1 that circulate in the
population throughout the year follow a similar seasonality as the other
common respiratory agents peaking in the winter months but without the 2-
year rhythm. It has also been observed that CoVs are frequent as co-infecting
pathogens [37].
RSV is the predominant cause of bronchiolitis and pneumonia in infants
below 6 months of age [12, 38], a condition that frequently requires
hospitalization due to respiratory failure [7]. Approximately 70% of all
children have had an RSV infection during their first year, and nearly all
children have experienced one or several RSV infections in their second year
of life [39]. Children born prematurely or with underlying disease such as
bronchopulmonary dysplasia, congenital heart disease or neuromuscular
disease are at higher risk of severe respiratory disease following RSV
infection [40-42]. In addition to infants and small children, RSV can also
affect adults with the greatest disease severity in elderly and immunodeficient
individuals of any age [43, 44]. The host-to-host spread of RSV occurs
through the contact with virus-containing respiratory secretions in the form of
large aerosol droplets or through contaminated material that reach the
mucosal surface of nose, mouth or eye [45]. The primary RSV infection
usually concerns the ciliated cells of the nasal cavity, and the first symptoms
of RSV disease normally appear at 4-5 days after initial infection [46].
From the nasal cavity the virus can spread to the lower respiratory tract and
infect ciliated cells of the bronchi and the type II lung alveolar cells, where it
recruits immune cells. Since a Th1 cell immune response, predominant in
virus infections, is not fully mature in infants under the age of 6 months, it is
frequent that RSV stimulates the Th2 response typical for parasites and
allergic diseases resulting in airway hypersensitivity due to production of
specific cytokines, IgE, and infiltration of eosinophils [Reviewed in 47]. The
progression of RSV infection to the bronchial epithelial cells and the
concomitant Th2 immune response results in dysfunction and subsequent
damage of the mucus-transporting ciliated cells [48, 49] as well as necrosis of
the bronchial epithelium. The extensive infiltration of inflammatory cells into
the respiratory epithelium in combination with excessive mucus production,
dysfunctional mucus transport and increased proportion of dead cells can lead
to airway obstruction and progression into pneumonia. In severe cases, the
obstruction can cause emphysema and/or collapse of the small distal airways
[48, 50]. This becomes particularly evident in infants and young children
where the lower airways are very narrow and vulnerable to obstruction [13].
Candidate antivirals for treatment of respiratory syncytial virus and coronavirus infections
4
Furthermore, given the substantial involvement of the allergic type Th2
immune response in RSV disease in infants, several studies indicate that the
risk of developing asthmatic and obstructive respiratory conditions is higher
following a severe RSV infection at an early age [39, 51, 52].
RSV produces a characteristic cytopathic effect upon infection of monolayers
of cultured cells, i.e., formation of syncytia [53], a feature contributing to the
name of this pathogen (Figure 1). However, the syncytium forming activity
of RSV is not usually observed when studying the infection in differentiated
cultures of respiratory epithelial cells, and even though it can be observed in
some RSV infected patients it is not a dominant feature [54, 55]. RSV
infection can also extend to other organs causing conditions like myocardial
damage, cardiac arrhythmias, neurological abnormalities and hepatitis.
Furthermore, the presence of virus or virus related material have also been
found in samples of peripheral blood, cerebrospinal fluid, myocardium and
liver [Reviewed in 56].
Figure 1. Monolayer cultures of HEp-2 cells infected with RSV and stained with crystal violet.
Uninfected cell monolayer (left image), the RSV-induced cytopathic effect in the form of
syncytial plaques (middle), and an enlargement of syncytium formed by fusion of many cells
reflected by presence of multiple nuclei seen as white spots (right image).
Human CoVs can cause respiratory disease of varying severity. Among the
six CoVs known to infect humans, the disease severity ranges from mild
common cold symptoms to severe acute respiratory disease with potential
fatal outcome. The human infection with CoV causing a mild common cold-
like disease, i.e., 229E, OC43, NL63 and HKU1 is usually limited to
epithelial cells of the nasal cavity where these viruses infects non-ciliated
secretory cells (229E) or ciliated cells (OC43, NL-63, HKU1) [57]. Infections
caused by the common CoVs 229E and OC43 generally include symptoms
such as fever, cough, malaise, headache and sore throat that last for
approximately 3-4 days but are often self-limiting [58, 59]. However, these
strains as well as CoVs NL-63 and HKU1 have also been reported to cause
Anna Lundin
5
more severe upper and lower respiratory infections in both children and
adults with symptoms of bronchiolitis and pneumonia [60, 61]. SARS-CoV
exhibits a more severe course of infection leading to hospitalization in 20-
30% of the cases [27] with a mortality rate of approximately 15% [62].
SARS-CoV predominantly causes pneumonia with generally less symptoms
from the upper respiratory tract than in the other CoV infections, and with
presence of non-respiratory symptoms such as diarrhea [27, 63]. Analysis of
deceased patients indicated the involvement of multiple organs such as the
lymph nodes, heart, liver and kidneys [64, 65]. Like many other respiratory
viruses CoVs spread through infected respiratory secretions or fomites [58,
66].
Candidate antivirals for treatment of respiratory syncytial virus and coronavirus infections
6
Human RSV belongs to the family of Paramyxoviridae of the order
Mononegavirales that groups viruses such as parainfluenza, mumps and
measles containing a single nonsegmented negative sense RNA genome.
More specifically, pneumotropic RSV together with human MPV and their
animal counterparts are classified within the subfamily of Pneumovirinae.
Figure 2. The RSV virion. (A) Cartoon structure of the viral particle showing presence of the
F-fusion-, G- attachment, and SH-small hydrophobic glycoproteins protruding from the virion
lipid envelope; and the M-membrane scaffolding protein, and the negative-sense single strand
RNA genome associated with the N-nucleocapsid, P-phosphoprotein, and the L-polymerase.
(B-C) EM images of RSV particles in the spherical (B) and filamentous (C) forms.
RSV is an enveloped pathogen that contains a genome of approximately 15
kb embedded into a nucleocapsid with a symmetric helical, “herring-bone”
formation [67]. The genome carries 10 genes that encode for 11 proteins in
the following 3’ order: nonstructural proteins 1 and 2 (NS1, NS2),
nucleocapsid-, phospho-, and matrix proteins (N, P, M), surface
Anna Lundin
7
glycoproteins (SH, G, F), protein M2, consisting of M2-1 and M2-2, and
polymerase (L) protein. The M2 gene which partly overlaps the L protein
gene is encoded from two overlapping open reading frames [68]. The RSV
virions (Figure 2A) are pleomorphic and their shapes vary from a range of
rounded semi-spherical forms of about 80-350 nm in diameter (Figure 2B) to
the most prevalent long extended filament form with a diameter of 60-200
nm extending up to 10µm in length (Figure 2C). The budding of viral
filaments from cells is frequently incomplete and these virions remain
associated with the cell surface thus resembling cellular microvilli [69, 70].
RSV virions possess three kinds of structural glycoproteins, known as G, F,
and SH, that are embedded in the viral envelope and occur in form of surface-
projecting spikes. While the biological function of the SH-protein in the RSV
life cycle still requires further clarification [71], the G- and F-glycoproteins
contribute essential functions in the RSV attachment and fusion events.
The initial interaction of RSV virions with susceptible cells occurs through
binding to cell surface glycosaminoglycan (GAG) chains, a step mediated by
the viral G-protein [72] discussed further in section 1.4.3, (Figure 11). The G
component is a type II glycoprotein of 289-299 amino acids (aa) with a
hydrophobic membrane-spanning domain at the N-terminal end that serves as
a signal sequence and anchors the protein to the virion envelope [73, 74]. The
ectodomain of the protein contains a central cysteine rich region (aa 173-186)
flanked by the C-terminal positively charged GAG-binding region (aa 187-
217) [75, 76] and the N-terminal stretch of negatively charged and
hydrophobic aa at positions 160-172. Residues at positions 164-176 that
partly overlap the cysteine rich region and the negatively charged domain are
highly conserved among different strains of RSV. The central domain is
flanked by two mucin-like regions which are referred to as such due to the
high content of aa serine, threonine and proline in addition to heavy O-
glycosylation that resembles similar structural features of mucins found at the
surface of epithelial cells.
Candidate antivirals for treatment of respiratory syncytial virus and coronavirus infections
8
Figure 3. Schematic structure of the RSV G-protein showing sequential order of essential
regions. The aa sequence of the central region including the cysteine noose induced by cross-
linking of cysteines at positions 173 and 186, and 176 and 182 are shown in red. Other
regions, namely the negatively charged, the conserved, the superantigen, and the GAG-
binding regions and their aa span are also indicated. CT, cytoplasmic tail endodomain; TM,
N-terminal transmembrane domain; MLR, mucin-like region; CCCC, cysteine nose; GAG-BD,
GAG-binding domain.
The mucin-like regions of the G-protein are highly variable in sequence,
which contribute to the G-protein being the RSV gene product with the
highest sequence variability between different strains [73, 77]. Both O- and
N-linked glycans constitute a substantial part of the mass of the G-protein
which is synthesized as a fairly small protein precursor (32 kDa), and further
modified by co-translational addition of N-linked sugars a n d subsequent O-
glycosylation in the trans-Golgi compartments [78]. The presence of serine
and threonine residues accounts for approximately 30.6% of the total aa
content which infers presence of over 70 possible sites for O-linked
glycosylation [79]. Since the mature form of the G-protein is approximately
80-90 kDa in size, the glycans contribute to approximately 60% of its
molecular mass [80-82]. The mucin structures of the G-protein may stretch
out the protein, shade and protect the central region and its antigenic sites
from immune response or proteases, and make the protein hydrophilic and
sticky.
The cysteine rich region of the G-protein is conserved and contains four
cysteine residues (Figure 3) at positions 173, 176, 182 and 186 (in A2 strain).
These cysteines are linked by two disulfide bridges between residues 173-186
and 176-182 respectively forming a mini-loop or “cysteine noose” [83-85].
The major function of this structure is likely to provide a tension to
sequences that flank the cysteine noose, i.e., the GAG-binding and the
negatively charged regions, a feature that could facilitate interaction of these
Anna Lundin
9
regions thus modulating the virus binding to or its removal from the cellular
GAG receptors (Figure 11). Variations in charges, exposed residues and
regulation of protein folding in these three central domains of the G-protein
are likely to confer specificity in receptor binding [82]. Furthermore, based
on the conserved nature of the cysteine rich region, this structure is also an
important antigenic site in RSV of both human and animal origin [84, 85].
Apart from the virion envelope associated G-protein, RSV also expresses a
truncated, secreted form of this component (Gs), where approximately 65 aa
of the N-terminal end, including the CT and TM regions, are trimmed off.
This Gs form accounts for about 20% of the total amount of G-protein
produced, however this value may rise to approximately 80% during the first
24 h of RSV infection [86].
Gs have several suggested roles in interference with the immune response. In
particular, (i) Gs may act as decoy for the host antiviral defenses including
trapping of the virus-neutralizing antibodies [87]. (ii) It may impair innate
immunity by inhibiting signaling from the Toll-like receptors (TLR) 2, 4, and
9 important in recognition of the viral structural envelope proteins. (iii) The
central region of the G-protein possesses CX3C fractalkine-like motifs that
may hamper the cellular Th1 immune response, and (iv) the central portion of
the G-protein overlapping part of the cysteine noose and the GAG-binding
region, i.e. the stretch 183-WAICKRIPNKKPGKKT-198 (Figure 3) [75, 76,
88] is suggested to be a superantigen that provokes hypersensitivity of
airways by stimulating Th2 immune response typical for allergic diseases
[68, 89, Reviewed in 90]
Attachment of RSV particles to cells, an event mediated by the G-protein, is
prerequisite for the second step of the viral life cycle, i.e., its penetration
through the cell plasma membrane into the cytoplasm. This event is mediated
by the specialized fusion device of the F-protein that shows structural and
functional resemblance to a wide range of other virus fusion proteins [91-94]
including these of other paramyxoviruses [95]. It has been shown that the F-
protein can be sufficient for all the necessary roles in RSV attachment, entry
and fusion processes and hence the virus may cope without the presence of
the G or SH proteins. However, in spite of the successful replication in cell
culture of a mutant virus lacking G and SH, the virus infectivity was
attenuated and inefficient [96, 97].
The RSV F-protein, a type I glycoprotein comprising 754 aa, is mainly
responsible for the fusion between the lipids of viral envelope and the cell
plasma membrane, but also for the cell-cell fusion activities during the virus
Candidate antivirals for treatment of respiratory syncytial virus and coronavirus infections
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spread [98] manifested as the formation of characteristic syncytia of infected
cultured cells (Figure 1, section 1.1.1) [53]. The F-protein (Figure 4) is
synthesized as a single polypeptide precursor, F0, which is co-translationally
glycosylated in the ER and subsequently cleaved in the Golgi compartments
by the furin-like cellular endoproteases. This yields the two disulfide-linked
subunits, F1 and F2, which represent the biologically active form of the F-
protein inserted into the budding membrane of progeny RSV virions [99,
100]. The N-terminally located F2 subunit is smaller than the F1, it holds the
signal peptide, possesses four potential sites for N-glycosylation [101], and is
believed to have the receptor-binding activities [102]. The larger F1 subunit
is a specialized fusion device and comprises an N-terminal fusion peptide
hidden in the pre-fusion form of the protein [95, 103, 104], followed by the
heptad repeat 1 (HR1), an intervening domain of over 250 aa [105], the
heptad repeat 2 (HR2), transmembrane domain (TM), and a short
cytoplasmic region [101]. Since the F-protein forms trimeric spikes in viral
particles, the HR1 and HR2 fold into trimeric coiled coil structures where the
α-helices are organized into an antiparallel heterodimer so that the
hydrophobic surfaces face each other [106].
Figure 4. Schematic structure of the disulfide linked F1 and F2 subunits of the RSV F-protein
showing sequential order of the FP-fusion peptide, HR1/HR2-heptad repeats, CR-
central/intermediate domain, TMR- transmembrane region, and the CT- cytoplasmic tail.
How does the F1 protein mediate fusion between lipids of the viral and
cellular membranes? The binding of the RSV particle to a host cell, an event
that can be mediated by the G-protein or the F2 subunit, triggers three major
changes in conformation of the prefusion “cone”-like form of the F-protein
[91, 107-109] (Figure 5). First, the F1 subunit acquires an extended
intermediate shape as a result of exposure of its N-terminal
hydrophobic/lipophilic fusion peptide that, as mentioned above, is hidden in
the prefusion form of the protein [95, 104]. The viral fusion peptide is
immediately inserted into lipids of the adjacent cell plasma membrane so the
virus particle and the cell are strongly bound through the F1 subunit which is
anchored in the viral lipid envelope by the TM region. The next step in the
virus-cell fusion is mediated by the HR1 domain which is located close to the
N-terminal fusion peptide inserted into the cell, and HR2 domain that lies
close to the C-terminal TM region inserted into the viral envelope. As
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mentioned above the HR structures are in fact trimeric α-helices that
automatically fold into the coiled coil structure. Insertion of the fusion
peptide into the cell plasma membrane triggers the movement of HR1 and
HR2 towards each other concluding in their automatic folding into a
hexameric complex, also referred to as a six helix bundle. This
conformational change is paralleled by alteration in the shape of the protein
from an extended to a collapsed or hairpin intermediate. An obvious outcome
of the HR1 and HR2 interaction is a tight apposition of viral and cellular
membranes. This event distorts the membranes and promotes the hemifusion
between the outer bilayers, followed by complete fusion, pore formation in
the fused membrane and insertion of viral ribonucleocapsids into the
cytoplasm [103, 107]. The basic hairpin structure of the F-protein of RSV
and other paramyxoviruses is related to that of other viruses such as SARS,
HIV and influenza [105] and these similarities suggest that the fusion process
is a conserved mechanism [110].
Figure 5. Schematic representation of the RSV F-protein mediated membrane fusion. (A) In
the native prefusion form the trimeric F-protein is inserted in the viral envelope with its fusion
peptide (in blue) hidden inside the protein. The RSV binding to cells triggers changes in the
conformation of the F-protein resulting in (B) protein elongation, and exposure and insertion
of fusion peptide into cellular membrane. (C-D) Owing to the strong affinity that the two
heptad repeat regions (in green and pink) have for each other, the protein collapses forcibly
into the six helix bundle conformation, thus apposing the viral and cellular membranes, and
(E) inducing their fusion.
During the virus-cell fusion the F-protein undergoes a series of
conformational changes resulting in several intermediate forms, and a final
postfusion “lollipop” shape [109]. All these forms of the F-protein are
attractive targets for antiviral development, and in fact numerous drug
Candidate antivirals for treatment of respiratory syncytial virus and coronavirus infections
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candidates for RSV treatment are of the fusion inhibitor class (discussed
more in section 1:6). Presence of the F-protein intermediates is also important
for the vaccine development. Several antigenic sites located in the
intervening domain between two HR regions of the F-protein, mainly in the
central cysteine rich region near the HR2 were identified [111-113].
Structural analysis of the F-protein using EM-analysis revealed two different
shapes resembling a “cone” (prefusion) and a “lollipop” (postfusion) with the
location of the antigenic sites grouped on the “head” region of these protein
structures [109].
Following insertion of viral ribonucleocapsid into the cell, further processing
takes places entirely in the cytoplasm, more specifically in the viral
replication sites known as inclusion bodies. These RSV-induced structures
are not well characterized. They are clearly visible in light and electron
microscope as regions of electron dense cytoplasm with several internal
vacuoles and membrane-containing adjacent vesicles [114]. The viral RNA
delivered to cytoplasm is specifically covered by the N protein, the aim being
to protect RNA from recognition by the cellular interferon system but permit
specific activity of the viral L polymerase [74, 115]. The polymerase
complex proteins, i.e., the L-protein as the main RNA-dependent RNA
polymerase (RdRp) in addition to the P, N, M2-1 and M2-2 constitute the
core RNA replication system in RSV [116]. The phosphoprotein (P) binds to
both the L polymerase and to the N protein on the ribonucleocapsid to
transiently uncover the small segments of RNA thus helping the polymerase
in specific recognition of viral RNA [Reviewed in 90, 117, 118].
Furthermore, the interaction of P-protein with M2-1 is important for its
proper function in elongation of the newly formed RNAs [116, 119]. Later
during infection the RNA transcription shifts over to replication, an event
suggested to be regulated by the M2-2 protein [74]. This shift results in the
generation of a full length cRNA or antigenome with opposite polarity, which
is used as a template for new full length genomic RNA to be incorporated
into progeny virions [68, 74]. The sequentially transcribed mRNAs are then
translated into the following viral protein products NS1, NS2, N, P, M, SH,
G, F, M2 (M2-1 and M2-2) and L. The N, P, M and F-proteins are required
for the formation of new virus particles [120]. The nonstructural proteins
NS1 and NS2 are unique to the RSV family and have no counterpart in other
members of the pneumovirinae [121]. NS1 and, to a lesser extent, NS2 are
known to inhibit the induction of interferon [122, 123].
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Following production of progeny viral RNA and proteins an assembly of
these components takes place. The M protein is critical in this event since it
mediates the association of the nucleocapsid with the budding viral envelope
mainly due to interaction with both the viral nucleocapsid and the F-protein
of the viral envelope, a feature of importance for the morphogenesis and
formation of new virus particles [120]. The M-protein is transported to the
plasma membrane and partly incorporates into lipid rafts, which are the
suggested sites for assembly and budding of progeny virions [124, 125].
Proteins and new genomic RNA assemble close to the membrane surface
where the structural proteins are incorporated into budding host membranes
after processing in the ER/Golgi [99]. The M- and the F-proteins
incorporated in the lipid rafts are associated with cytoskeleton components
such as actin and microtubule which in turn is significant for virus assembly
and release [120, 126] and/or the formation of the filamentous viral particles
associated with the cell surface [127].
Candidate antivirals for treatment of respiratory syncytial virus and coronavirus infections
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Human respiratory CoVs, i.e., 229E, OC43, NL63, HKU1, SARS-CoV and
MERS-CoV belong to the family of Coronaviridae in the order Nidovirales,
and hold the largest genome of all known RNA viruses spanning between 27-
32kb. CoVs are enveloped pathogens with a non-segmented positive sense
single strand RNA genome associated with a nucleocapsid protein in a helical
formation. The virus particle is mainly spherical of ~100-160 nm in diameter
[128] (Figure 6).
Figure 6. Structure of the CoV virion. (A) Cartoon showing presence of the large protruding
spike (S), membrane (M) and envelope (E) proteins that provide a scaffold for the viral
envelope, and nucleocapsid (N) protein associated with a single strand viral RNA. (B) EM-
images of a single CoV particle (top) and cluster of CoV particles (bottom).
Since CoVs contain a positive sense RNA that could be directly translated by
cellular ribosomal machinery there is no need for the presence of RNA
replicative enzymes in the virions. Therefore, most CoVs hold a set of only
four structural proteins, i.e., the spike (S), envelope (E), membrane/matrix
(M) and nucleocapsid (N) protein while some human CoVs such as OC43
also carry an additional haemagglutinin esterase (HE) protein [128].
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The S-protein is a large (~200 kDa) glycoprotein protruding from the surface
of the virus envelope giving it a characteristic crown (=corona)-like structure.
The S-protein is heavily glycosylated and is composed of two large subunits,
S1 and S2, that mediate the virus attachment to and entry into the susceptible
cells respectively [129, 130]. CoVs can penetrate into the cells by direct
fusion between lipids of the viral envelope and the cell plasma membrane or
through the membrane of endocytic vesicles (e.g. SARS-CoV) triggered by
their acidic environment. The mechanism of the S2-induced fusion is similar
to that caused by the F-protein of RSV, and results in the delivery of a core
structure, i.e., a complex of viral RNA with the N protein, into the cytoplasm
where the entire replication process occurs. The N protein, apart from
important roles in package, condensation and transcription of genomic RNA,
also influences pathogenesis of CoV infections [131, 132].
Following dissociation of the nucleocapsid core, a naked viral RNA is
released. As mentioned above, since CoVs have a positive strand genome it
can act directly as an mRNA template for translation of proteins in
ribosomes. Approximately two-thirds of CoV genome is devoted to the
production of 16 viral non-structural proteins (nsps). Some of these nsps
recruit and modify cellular lipid membranes to produce sites for replication of
viral RNA while the other nsps which carry a range of activities such as
polymerase, helicase, exoribonuclease, endoribonuclease and
methyltransferase, perform and/or regulate replication of viral RNA (Figure
7). Since nsps of CoVs are the matter of this thesis (see paper IV) some
aspects of their activities are further described in section 1.3.3.
The genomic RNA is also used to generate a new minus strand RNA for
further synthesis of full length genome and for production of subgenomic
mRNA. The structural proteins S, E, M and N are translated from
subgenomic mRNAs, undergo processing in the Golgi and are subsequently
incorporated into membranes and/or transported to the site of assembly at the
ER and Golgi compartments [133, 134]. As mentioned above the N-protein
binds to newly synthesized RNA forming the nucleocapsid structure which is
transported to the site of assembly [132, 135]. The viral M protein, the most
abundant CoV structural protein, is critical for this process. It is fairly small
(~10kDa) and possesses three transmembrane domains. The protein main
activity is in virion assembly and morphogenesis [136], and it performs this
task by interaction with the S-protein of the viral envelope and the N-protein
of the nucleocapsid. This activity helps to organize the virus budding, to
Candidate antivirals for treatment of respiratory syncytial virus and coronavirus infections
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incorporate spikes into the viral envelope, and to package the nucleocapsid
into virions [137-140]. CoV virions bud from the ERGIC [134, 141] where
the activities of the M and another envelope component, the E protein, are
required in formation of envelope curvature of progeny virions as well as in
pinching the budding virions off the membrane surface [137, 142, 143].
Progeny viruses accumulate in large exocytic vesicles to be transported
towards the plasma membrane and subsequently released.
An interesting feature of CoVs, that has yet to be fully resolved, is the
mechanism of their replication employing the large set of 16 nsps in the
intricate replication machinery characteristic for this family of viruses. The
number of encoded nsps and their involvement in replication of RNA is
unusual among all RNA viruses, and therefore CoVs uphold the largest RNA
genome known to date. As mentioned earlier these 16 nsps are derived from
the proteolytic cleavage of two large polyproteins 1a and 1 ab. The cleavage
is mediated by three different proteases which are included in the
polyproteins and need to be liberated by autocatalysis to cleave the rest of the
polyprotein. These include the papain-like cysteine proteases PL1pro and
PL2pro, located in nsp3 [144], and the chymotrypsin like main protease
3CLpro, located in nsp5. The cleavage products of these proteases and their
putative functions in replication of CoV RNA are shown in Figure 7.
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Figure 7. Putative biological functions of the 16 nonstructural proteins (nsps) encoded by
CoVs. Genomic organization of sequences coding for these 16 nsps and 4 structural proteins is
also shown. RC-replication complex; RdRp, RNA-dependent RNA-polymerase.
As seen from Figure 7, nsps 7-16 are directly involved in replication of viral
RNA including its processing, proofreading, and regulation. The
proofreading capacity of the nsp14 3’-5’ exoribonuclease have a key function
in upholding an unusually high replication fidelity of CoV and contribute to
the uniquely large genomes of these RNA viruses [145, 146]. Nsp5 and part
of nsp3 are proteases, while nsp3, nsp4, and nsp6, all of which are
transmembrane proteins, contribute to the formation of CoV replication sites
or “viral organelles” in the cytoplasm by recruiting and modifying the host
cellular membranes. Nsp3 and nsp4 are believed to pair recruited membranes
to form convoluted membranes (CM) while nsp6 is thought to form a set of
single membrane vesicles. Concert actions of these three proteins [147] result
in formation of the reticulovesicular network of paired, closely apposed
membranes (Figure 8A) that includes CM, double membrane vesicles (DVM)
(Figure 8C-D), and vesicle packets (VPs) all connected to ER through the
outer membrane. The ~200-300 nm vesicles may appear as early as 2 h p.i.
and continuously change in number and in size throughout the infection [148-
151]. The CM structures are also believed to be sites for accumulation of the
RNA replication complex subunits including among others nsp8 and nsp12.
VP structures are formed later during infection and frequently contain
multiple inner vesicles as well as large numbers of budding or fully
assembled virions (Figure 8E) indicating that VPs might be created by DMV
merger [150]. Although not fully resolved, the main roles of these structures
during CoV replication are believed to be as follows.
Candidate antivirals for treatment of respiratory syncytial virus and coronavirus infections
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(i) Assembly and concentration of replication components. The wide
range of activities within the 16 nsps of the replicase machinery needs to
be organized and concentrated for optimal function in a confined space of
the modified membrane structures [152]. In addition, retaining the
negative strand RNAs (an antigenome of CoV) within a limited space
may increase the template specificity [153].
(ii) Structural framework and foundation for membrane anchoring of the
replication-transcription complex (RTC) of nsps. Many of the nsps
especially those that possess TM region(s) such as nsp3, nsp4, and nsp6
have a suggested scaffolding/membrane anchoring functions for the rest
of RTC nsps. Furthermore, the enzyme activities for some of the
subcomponents of the RTC have been shown to require tethering for
optimal efficiency [144, 152] an observation that fits well to the theory of
immobilized enzymes and moving templates in replication/transcription
systems [154].
(iii) Protection. The double stranded RNA intermediate that occurs
during virus replication is a very strong signal of a presence of a “foreign
body” in the cells [155]. Restricting the viral replication process to the
inside environment of membrane compartments delays the detection by
antiviral host cell responses (interferon system) and shield the production
of new genetic material. Thus, the double strand RNA intermediate
product of CoV replication is hidden inside the internal vesicle of the
DVM structure [150].
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Figure 8. (A) Schematic model of the reticulovesicular network of modified ER membranes
that support CoV RNA synthesis (Figure adapted from Knoops et al PLoS Biology, 2008). (C-
E) EM-images of the 229E-CoV induced clusters of DMVs (arrows) and viral particles found
in MRC5 cells at 18 h p.i. Note the lack of these structures in uninfected cells (B). CM,
convoluted membranes; ER, endoplasmic reticulum; DMVs, double membrane vesicles; VPs,
vesicle packets.
Candidate antivirals for treatment of respiratory syncytial virus and coronavirus infections
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The conducting respiratory airways comprise the tissues that line the nasal
cavity, pharynx, larynx and trachea down to the branched bronchial tree and
the small alveoli. The tissues of the human airways constitute a protective
barrier between the outside and internal environments of the body. In
addition, the epithelial tissues of the respiratory tract comprise properties that
extend far beyond having “just” a barrier function. Except for the normal
regulatory and metabolic functions the epithelium has multiple roles in
protection and defense of the respiratory system such as clearing out potential
inhaled agents, attracting and activating inflammatory cells, and recruiting
immune cells. The respiratory tract is lined with pseudostratified columnar
epithelium in the nasal cavity, larynx, trachea and bronchi, and simple
squamous epithelium in the small alveoli.
The major constituents of the respiratory epithelium are these schematically
shown in Figure 9. The basal cells are attached to the basement membrane
and anchor the superior columnar cells that can be either ciliated or secretory
cells. The ciliated cells are located at the surface of the epithelium and
possess up to 300 cilia/cell. Since the primary function of these cells is to
transport airway fluids together with any trapped pathogens towards the nasal
cavity and oropharynx via the coordinated beating of the cilia, there is an
abundance of mitochondria in the cells providing the necessary energy for
this process [156]. The major types of secretory cells in the respiratory
epithelium include, mucus/goblet, clara, and serous cells that produce and
secrete large amounts of fluids that are important for proper airway function.
Mucus/goblet cells produce mucins, a major constituent of respiratory fluid
covering the epithelium. Clara cells are believed to produce bronchiolar
surfactants and protease inhibitors, and can be found mainly in the smaller
airways of the bronchi.
Respiratory mucus is a viscous fluid that covers the surface of the epithelium
and is produced by the different secretory cells mentioned above. The fluid
contains a range of molecules that is carefully balanced to maintain
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physiochemical properties required for function and protection of the
epithelium and for facilitated transportation towards the nasal cavity and
oropharynx. The viscosity of the mucus layer decreases in parallel with the
reduction in the size of the airways [157, 158].
The major constituents of mucus are secreted mucins that form a network
through extensive coupling of cysteine disulfide bridges. The mucins are
elongated and heavily O-glycosylated proteins where the sugar entities
account for almost 70-80% of the mucin weight [159]. Differences in the
glycosylation patterns create a wide range of mucin variants that help to
produce the mucin network optimal for the proper physiological and
defensive function of the mucus layer [160]. The O-linked glycans of mucins
are highly hydrophilic and their capability to bind water and ion molecules
contribute to mucous viscosity [161]. The glycan chains are often terminated
with negatively charged sialic acid or less frequently with sulfated
monosaccharide residues [159, 162] which in addition to providing receptor
sites for different pathogens [163] may contribute to the acidity of the mucin
granules secreted by mucus/goblet cells, and may regulate the viscosity of the
secretions [156]. The viscous mucin layer has no direct contact with the
epithelial cells, and they are separated by a less viscous layer of fluid that
permits undisturbed beating of cilia and thereby an easy transportation of
mucus.
Figure 9. Schematic structure of the airway epithelium. Note the exclusive expression of APN
by goblet and ACE-2 receptor by ciliated cells, a feature associated with specific tropism of
CoVs 229E and NL-63 respectively. GAGs, glycosaminoglycans; APN, aminopeptidase-N;
ACE-2, angiotensin converting enzyme 2. Illustration kindly prepared by Anders Lundin.
Candidate antivirals for treatment of respiratory syncytial virus and coronavirus infections
22
The mucus layer contains multiple substances with antimicrobial properties.
Surfactant proteins A and D have essential roles in the airway defense
including direct anti-infectious activities as well as modulation of
inflammatory response by the induction of cytokines [164]. Furthermore,
mucus contains a wide range of proteolytic enzymes, antiproteases,
antimicrobial peptides, and antimicrobial proteins such as lactoferrin,
defensins or lactoperoxidase, known for their antiviral properties [Reviewed
in 165]. Moreover, respiratory secretions also contain a large number of
antimicrobial lipids and sterols such as free fatty acids, cholesterol, and
cholesteryl esters that contribute to the airway barrier defense function [166].
The main function of the cilia is to transport mucus towards the oropharynx
with subsequent movement to the stomach. This is an important feature of the
defense system where pathogens, toxins and various particles are captured in
the mucus and later destroyed in the acidic environment of the stomach. As
mentioned above an optimal viscosity of the mucus and an undisturbed
strength and movement of cilia beating are crucial for mucus transportation
and the defense against pathogens. Infection of the ciliated cells with RSV,
CoVs and other viruses can cause both functional impairment manifested as
decreased frequency and in some cases dyssynchronization of ciliary beating,
and morphological alterations such as loss of cilia-, and/or cell death and
their release into the mucus (Figure 10) [49, 167, 168]. Furthermore, RSV
has also been observed to utilize the beating cilia to spread to neighboring
ciliated cells [54].
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Figure 10. Morphological and functional impairment of the airway epithelial cells following
virus infection. These, among others, may include dyssynchronization of cilia beating, loss of
cilia or death and fragmentation of ciliated cells, and thickening of the mucus layer that
collectively results in impaired transport of mucus. Illustration kindly prepared by Anders
Lundin.
The different proteins and/or carbohydrate components located on the
surfaces of ciliated/secretory cells can provide receptor sites for initial
interaction of respiratory viruses. The distribution of these proteins on a
particular cell type and/or at specific parts of the respiratory epithelium, may
contribute both to the viral tropism and therefore pathogenesis of infection
(Figure 9) [54, 169].
RSV specifically targets ciliated cells of the respiratory epithelium and is
capable to spread to all parts of the conducting airways. The infection is
polarized with both initial infection and budding of progeny virions taking
place at the apical surface of the epithelium [54, 170]. The identification of a
“true” cellular receptor molecule for RSV has been proven to be a challenge
and although a number of candidate receptors have been suggested the issue
still remains to be fully resolved.
An example is nucleolin, a protein associated with cell proliferation and
growth [171], and recently suggested as being a functional receptor for RSV
Candidate antivirals for treatment of respiratory syncytial virus and coronavirus infections
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[172]. Although nucleolin is most abundantly expressed in the nucleus [173]
it can also be found on the cell surface as a part of a protein complex [174].
However, nucleolin is expressed in most cell types and therefore unlikely to
contribute to specific tropism of RSV [172].
One class of molecules identified as initial binding sites for RSV are cell
surface GAGs. Structurally, these molecules are large linear chains of
repeating disaccharide units which are heavily sulfated and hence have a
strong negative charge. Several different GAG classes exist which differ in
the type of amino sugar, uronic acid component, glycosidic linkage, and
sulfation pattern. Chondroitin sulfate, dermatan sulfate, keratan sulfate (KS),
heparin and heparan sulfate (HS) represent the major classes of sulfated
GAGs [175]. The GAGs are abundantly expressed on the surface of nearly all
animal cells. Their function in binding and regulating activity of a plethora of
different proteins is crucial and involves interactions with proteins such as
growth factors, enzymes, and chemokines [176, 177]. GAGs have been
shown to provide attachment sites for a range of different viruses such as
herpesviruses, HIV, flaviviruses and alphaviruses [178-181]. Several studies
have emphasized the importance of GAGs heparan sulfate (HS), chondroitin
sulfate and heparin for RSV infection in cell culture [76, 182]. However, HS
and chondroitin sulfate are poorly expressed on the apical surface of ciliated
airway epithelial cells [183, 184]. Instead, based on the suggested preference
of RSV for ciliated cells, GAGs such as KS, which have been shown to be
extensively expressed on these cells [54], may be important for mediating the
attachment of RSV in airway tissue.
The high negative charge of the GAG chains contributes to the multiple
electrostatic interactions with target proteins [175]. In RSV the major GAG-
binding component is the G-protein (Figure 11). A region in the G-protein
ectodomain, referred to as the heparin-binding domain (HBD) or the GAG-
binding domain, contains a sequence with a high content of positively
charged aa such as lysine or arginine. These basic aa residues are believed to
be involved in multiple electrostatic interactions with the negatively charged
sulfate/carboxylate groups of GAG chains thus mediating the initial binding
of RSV [75]. The RSV-GAG interaction should not be regarded as simple
charge association since specific distribution of basic aa in the GAG-binding
domain of the G-protein and the specific sulfation patterns of GAG chains
provide specificity for this interaction. [75, 185]. Even though GAG
interactions are important for RSV infection, it has been shown that mutant
viruses lacking the GAG-binding domain of the G-protein was capable of
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infecting mutant cell lines deficient in GAG expression [182, 186]. In
addition, RSV mutants deficient in expression of the G-protein can infect
cultured cells implying that the F-protein may also have the GAG-binding
properties or that there are alternative mechanisms that can promote the virus
attachment process.
Figure 11. Hypothetical model of the interaction of the RSV attachment protein G and cell
surface GAGs. (A) Central region of the G-protein is composed of a “relaxed” cysteine noose
flanked by the two oppositely charged regions i.e. the positively charged GAG-binding domain
and the amphipathic negatively charged domain which interact with each other. (B) The mucin
like region in the ectodomain of the G-protein may interact with mucins on the epithelial cell
surface, an event that brings the positively charged GAG-binding region of the G-protein into
close proximity to the negatively charged cell surface GAGs thus promoting their interaction.
This binding brings GAGs in closer to the negatively charged domain of the G-protein which
creates repulsion and tension in the cysteine noose, thus leading to liberation of the virion.
Candidate antivirals for treatment of respiratory syncytial virus and coronavirus infections
26
In contrast to RSV, the cellular receptor or co-receptor molecules for
respiratory CoVs have been identified. Many of these receptors and their
expression in different tissues have a clear connection to the viral
pathogenesis. In particular, aminopeptidase N (APN) and angiotensin
converting enzyme 2 (ACE2) act as functional receptors for 229E-CoV and
SARS-CoV respectively [187, 188]. Both proteins are abundantly expressed
in the airway epithelium consistent with predominant respiratory illness that
these viruses cause. In addition, both proteins can also be found in tissues
like the intestinal epithelium explaining why these viruses may also cause
enteric infections [64, 65, 189]. Like SARS-CoV, the NL-63-CoV utilizes
ACE 2 as its main receptor [190] while the 9-O-acetylated sialic acid [191]
and the major histocompatibility complex class I C (HLA-C) [192] molecules
were shown to facilitate infection of cells with OC43 and HKU1 CoVs
respectively. CoV tropism, strongly associated with exclusive expression of
specific receptors on certain cells, is exemplified by NL-63, OC43 and HKU1
which, similar to RSV, were found to target ciliated cells, while 229E mainly
targeted secretory/goblet cells. This tropism is dependent on the expression of
e.g. the CoV strain specific receptors ACE-2 and APN on ciliated and
secretory cells respectively (Figure 9) [57, 193].
Since the advent of cell culture techniques and their use for the virus
propagation and antiviral testing in the 1950s, there are now more than 50
antiviral drugs available for treatment of virus infections [194]. Most of them
are registered for treatment of HIV followed by those used against different
herpes- and influenza viruses [195]. In addition, recent advances in
development of antivirals for treatment of hepatitis C virus infection have
resulted in many new potential candidate drugs [Reviewed in 196]. Efficient
and non-toxic antiviral treatment against RSV and CoV infections is still
lacking at present, however new antiviral strategies for the inhibition of these
virus infections are continuously being developed (discussed further in
section 1.6).
Numerous strategies have been established for identifying and developing
new and effective antiviral drugs, one being the screening for antiviral
activity of large collections of compounds. This can be conducted either on a
broad, whole virus basis, which provides the possibility to identify new
targets for antiviral intervention or by more target specific screening looking
at the impact of a compound on a particular protein or enzyme activity
Anna Lundin
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(discussed further in section 1:7). Besides the screening approach other
strategies are focused on the particulars of the virus infection and interplay
with the host.
An important issue to consider when conducting antiviral testing in cell- or
tissue cultures is the possible adverse effect that the test compound in
question might have on cells. This issue is of special importance when the
compound can cause an unapparent cytotoxicity such as complete or partial
cytostatic effect and/or an altered morphology or metabolism of otherwise
viable cells. This may result in identification of “tricky” false positive
antiviral hits and their subjection to expensive evaluation in experimental
animals and clinics.
Since the virus life cycle is inevitably connected to a viable cell, all negative
impact of a potential antiviral on cellular components may affect its
replication in an unspecific manner. Notably, this mode of antiviral activity
of test compounds, i.e., targeting of cellular instead of viral components, is
acceptable provided that no adverse effects of potential antivirals on cells are
seen (see section 1:5:2). Hence, it is essential to use several different
cytotoxicity assays in antiviral discovery utilizing a live host assay system
that reflect various aspects of cell viability, proliferation, and metabolism
[197-200]. In an evaluation of antiviral potency of a hit compound it is
important to relate its safety to the antiviral efficacy. This is often assessed by
determining the selective index of the test compound, i.e. the range between
the lowest concentration that results in effective antiviral activity and the
highest concentration that does not cause cellular toxicity. This difference
determines the window of specific antiviral activity, also referred to as
“therapeutic index” in the clinical setting [201]. A compound with a narrow
selective index is difficult to evaluate in experimental animals and clinics,
and requires additional confirmation of antiviral potency and specificity since
the risk of a false positive hit is higher if the therapeutic index is low.
The viral life cycle involves a number of steps such as (i) attachment and
entry, (ii) uncoating and release of genetic material (iii) replication and
transcription (iv) virus particle assembly (v) and budding and/or release. Each
Candidate antivirals for treatment of respiratory syncytial virus and coronavirus infections
28
step includes components or their activities with potential for antiviral
targeting.
All viruses share the general features of the different life cycle steps
described below. However, every virus has its own particular way of
interacting with host cell components and metabolic pathways, largely
contributing to the difficulty of identifying a broad spectrum antiviral drug.
Described below and schematically illustrated in Figure 12 are examples of a
few of these strategies that resulted in the development of a
successful/approved antiviral drug.
Figure 12. Outline of the viral life cycle showing steps successfully targeted by antiviral
intervention. Specific events of the viral life cycle (purple) targeted by approved inhibitors
(green) are shown. TK, thymidine kinase; RT, reverse transcriptase; NRTIs, nucleoside analog
reverse-transcriptase inhibitor; NNRTIs, non-nucleoside reverse-transcriptase inhibitor.
Anna Lundin
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Targeting the steps of virus attachment to and entry into the cells is often
advantageous because the amount of virus present is usually quite low
allowing for a potent inhibitory activity that occurs prior to the initial virus
contact with cells [202]. The attachment and entry inhibitors can act through
several different inhibitory mechanisms including (i) binding to specific virus
attachment and entry proteins directly on the virion (ii) binding to the virus
receptor molecules at the surface of susceptible cells, and (iii) binding to the
intermediate, “activated”, form of a viral protein triggered in the attachment
or fusion process thus preventing further conformational changes necessary
to complete these steps.
In spite of potent antiviral activity in cultured cells many attachment/entry
inhibitors such as sulfated polysaccharides failed to protect humans in
clinical trials [203]. The first and perhaps the only drug approved is
enfuvirtide, a peptide derived from the HR2 region of HIV gp41 protein
targeting viral fusion activities and thus the entry of HIV into the cells
[Reviewed in 204].
The viral genetic material delivered into the cell is subjected to
replication/transcription events. Depending on the virus species, this involves
the viral and to varying extent the cellular machinery and may take place at
the nucleus, cytoplasm or both compartments. The genetic material of viruses
can be either DNA or RNA, and in the case of some RNA viruses an
intermediate DNA step may occur via the activity of reverse transcriptase
(RT). Because of the strict virus specificity of RT this enzyme is well suited
as an antiviral target, which led to the development of a new class of anti-
HIV drugs referred to as RT-inhibitors. These include both nucleoside- and
non-nucleoside analogs, NRTIs and NNRTIs respectively, which inhibit the
RT activity and act as chain terminators in DNA elongation [205, 206].
Another group of antivirals targeting the replication process are the DNA
polymerase inhibitors exemplified by the anti-herpesvirus drug acyclovir.
Acyclovir was the first highly selective and non-cytotoxic antiviral drug
discovered [207]. Being an analog of guanine, this drug also acts as a
premature chain terminator, similar to the RT-inhibitors for HIV, but its
specificity lies in a herpesviral enzyme, thymidine kinase (TK). To be
accessible to viral DNA polymerase, this drug must be enzymatically
activated (monophosphorylated) by TK. Acyclovir is suitable for enzymatic
activation by herpesviral TKs but not cellular TKs indicating that its activity
Candidate antivirals for treatment of respiratory syncytial virus and coronavirus infections
30
is restricted to the virus infected cells, a quality that makes this drug highly
specific with little or no cytotoxicity [207].
A classic example of an antiviral strategy targeting the virion release from
cells is the inhibition of influenza neuraminidase. Haemagglutinin (HA) is
the viral attachment protein that binds to sialic acid-containing entities on the
cell surface to mediate the virus binding to and subsequent penetration into
the cells. Although the sialic acid binding activity of influenza virus HA is
profitable for initial events in the viral life cycle, it is an obstacle in the virus
release stage since the progeny virions can be trapped at the cell surface and
cross-linked to form large clumps due to virus binding to sialic acid present
at the cell surface and on glycoproteins of other virions. At this stage, a
second influenza virus surface protein, neuraminidase (NA), cleaves the sialic
acid containing entity thus liberating trapped virions and releasing them from
the cell surface. The neuraminidase inhibitors, which are analogs of sialic
acid residue, exhibit tight binding to NA thus blocking the activity of this
enzyme and making it inaccessible to cellular sialic acid residues [208].
In addition to the virus specific targets, there are a number of different
intrinsic cellular features that are utilized by viruses such as those involved in
DNA, RNA and protein synthesis [205], in intracellular signaling, innate
immune mechanisms, and others. Therefore, features of the host cell
exploited by the virus can also be targeted by antivirals provided that such
intervention causes no adverse effects on cells. Targeting cellular processes
may have the advantage of a broader spectrum of inhibition since some
viruses use similar components during infection. Furthermore, antivirals
aiming at cellular targets tend to be less vulnerable to the development of
viral resistance. For example, ribavirin, a drug whose antiviral activity was
associated with “forcing” the viral polymerases to increase the number of
spontaneous errors/mutations to the level deadly for a virus, in the case of
RSV was shown to act as an inhibitor of cellular inosine monophosphate
dehydrogenase that depletes the cellular pool of guanosine required for
replication of viruses [209]. Furthermore, a wide range of intracellular
signaling pathways exemplified by RhoA used by RSV [210] and both NF-
kB and Raf/MEK/ERK signaling employed by influenza virus are promising
targets for antiviral intervention [Reviewed in 211].
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One of the greatest challenges in the development of antivirals is the
emergence of viral resistance. Viruses have an intrinsic property of high
speed replication and a remarkable capacity of adjusting to changes in their
environment. In addition, virus replication is evolutionary prone to errors
leading to a large number of virus variants, often called quasispecies, with a
higher chance of natural drug resistance. The rate of mutation and
recombination are the major determinants for emergence of drug resistant
viral variants, a powerful feature of viral replication leading to the selection
for resistant variants against practically all currently known antivirals [212].
The risk of resistance development is also increased in immunocompromised
individuals where the duration of infection is often extended or followed by
several consecutive infections requiring a prolonged drug exposure [213-
215].
Since the drug resistance is a major concern in the area of antiviral
intervention attempts to avoid, or at least reduce, the rate of this phenomenon
have been made. One such strategy is commonly applied in the treatment of
viral infections where several different classes of antivirals are available, as
in the case of HIV or HCV. Simultaneous challenge of the viral pathogen
with several drugs that target different viral components prevents the virus
from “finding” a mutational escape route other than lethal or at least a very
harmful combination of mutations for the virus. This can be exemplified by
the HAART therapy for HIV infection where the simultaneous treatment with
at least three antiviral drugs targeting the virus replication and transcription
by different mechanisms reduces the incidence of resistance [216]. Other
strategies focus on antiviral targets with available drug treatment towards
which resistant strains have already appeared. Here the design of a new
antiviral candidate with improved target interaction and more irreversible
impact could restore the drug sensitivity in previously resistant strains, and
decrease the risk for further development of resistance. An example of this
strategy is the development of a novel class of neuraminidase inhibitors for
treatment of influenza infections [217].
Candidate antivirals for treatment of respiratory syncytial virus and coronavirus infections
32
Early attempts to develop an RSV vaccine in 1966-1967 began with a
disaster when a formalin-inactivated RSV-vaccine failed to induce a
protective immune response resulting in a more severe illness upon natural
reinfection in vaccinated children than in the control group. This incidence
led to hesitations and large precautions in further development of RSV
vaccines [218, 219].
The reason of why the formalin-inactivated RSV vaccine enhanced the
disease is not completely understood, however, induction of inappropriate
cell mediated immunity i.e. an exaggerated allergy-like Th2 response has
been suggested. A Th2-biased response to RSV infection is associated with
increased levels of white blood cells in the lungs such as pulmonary
eosinophilia, in addition to IgE production and enhanced immunopathology.
In addition, the Th2 response is also associated with airway hyperreactivity
and increased production of mucus which can contribute to airway
obstruction. Moreover, the clearance of RSV infection can be delayed by a
high Th2 response since Th2 associated cytokines, such as IL-4 and IL-13,
are suggested to interfere with the virus clearance function of CD8+ T cells
[220, 221]. Furthermore, the antibody response induced by the vaccine was
altered in comparison to natural RSV infection and did not display an
efficient neutralization of RSV infectivity. This indicates that immunogenic
epitopes could have been damaged during vaccine preparation resulting in
insufficient protective immunity [222].
Although no RSV vaccine is currently approved, a prophylactic intervention
is available. Palivizumab, and its affinity-optimized variant Motavizumab,
are humanized monoclonal antibodies (Mab) specific for the RSV F-protein
[223, 224]. The antibodies are directed towards antigenic sites located in the
F1 subunit of the protein [225] and prevent the virus-induced fusion events
occurring between the viral and cellular membranes as well as in cell-to-cell
spread of RSV [226]. In the large clinical studies preceding the 1998 FDA
approval of palivizumab it was shown that prophylaxis with this Mab
resulted in a 55% reduction in the number of children needing hospital care in
comparison to control groups [227]. Palivizumab is administered by
intramuscular injections once per month during the RSV season at a dose of
15mg/kg. Even though the prophylaxis can reduce the incidence of
Anna Lundin
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hospitalizations the treatment is very expensive and therefore limited to
infants and children at high-risk of RSV infection, meaning mainly premature
infants or young children with underlying conditions such as congenital heart
disease or chronic lung disease [228]. Furthermore, the cost/effectiveness of
palivizumab administration is a matter of debate [229-231]
The only approved drug for treatment of RSV infection is the nucleoside-like
analog ribavirin. The drug is administered as an aerosol for an extended
period of time, and is usually provided by health care professionals in the
hospital setting. Ribavirin has a broad spectrum of antiviral inhibition for a
range of both DNA and RNA viruses [232, 233]. Several inhibitory
mechanisms have been reported for ribavirin and the specificity of its anti-
RSV activity is not entirely clear. The reported antiviral activity of ribavirin
includes inhibition of inosine 5’-monophosphate (IMP) dehydrogenase that
exhausts the guanine pool, blockade of the formation of the 5’cap on mRNA,
and inhibition of viral RdRp. In addition, ribavirin may also play a role in
enhancing antiviral immunity by shifting a Th2 response towards a Th1
response, and act as a mutagen pushing already high inaccuracy prone viral
RdRp over the “error catastrophe” edge [234, Reviewed in 235]. Even though
ribavirin is the only available treatment for severe RSV infection the
efficiency of this drug in RSV disease is ambiguous. The activity of ribavirin
is not specific for RSV and is considered to be suboptimal. In addition, the
treatment is fairly expensive and the drug has an uncertain safety profile
[236, 237]. Hence, due to the lack of effective and safe anti-RSV drugs,
numerous attempts at identification of novel drug candidates are being made.
Because of essential functions of the F-protein in the RSV life cycle, and the
presence of highly conserved structures, the fusion activity of this protein has
been selected as an important target for development of antivirals. Listed in
Table 1 are mostly small-molecule candidate anti-RSV drugs with antiviral
potency ranging from nanomolar down to picomolar concentrations as
exemplified by compounds BMS433771 and TMC-353121 respectively [238,
239]. During the virus-induced fusion the F-protein undergoes a series of
conformational changes and most fusion inhibitors interfere with and prevent
this protein from completing these transitions (Figure 13). The fusion
inhibitors target different areas of the F-protein such as the HR domains thus
preventing the formation of the six helix bundle complex (see section 1:2)
[238, 240]. It is suggested that the level of antiviral inhibition is dependent on
the degree of activation of the virus fusion protein. Meaning that if the
antiviral compound targets the activated, fusogenic, form of the protein the
Candidate antivirals for treatment of respiratory syncytial virus and coronavirus infections
34
antiviral potency depends on the rate of transition of the F-protein from the
inactive form into the active form [241].
Thus far, none of these compounds have progressed to late stage clinical
trials. Most compounds that have reached the early phase clinical testing are
later discontinued due to insufficient activity or undesirable safety- and/or
pharmacokinetic profiles. These compounds can display a very short time-
span of activity in vitro limited to the first hours of RSV life cycle [242].
However, recent reports of the optimized fusion inhibitor TMC353121
showed that the compound is active up to 48 hours after infection indicating
that a fusion inhibitor, although time-dependent, can have an extended
activity span likely caused by affecting the fusion events in the RSV cell-to-
cell spread [243].
Figure 13. Inhibitory mechanism of fusion inhibitors targeting the extended intermediate form
of the F-protein. The compound binds to the F-protein in its extended form (B) thus blocking
the interaction between the two HR regions (in green and purple) and/or stabilizing the
intermediate form in its extended conformation (relate C to D/E steps).
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Due to the relatively high variability in the attachment G-protein aa sequence
and, associated with it, decreased antigenic cross-reactivity between different
RSV strains, antivirals targeting this protein are far fewer than those affecting
the fusion process. Even so, the small-molecule candidate MBX-300
(NMSO3) composed of sulfated sialic acid conjugated to two alkyl chains,
was found to inhibit the RSV attachment to cells through interference with
the activities of the G-protein. The inhibitory effect occurred at low
micromolar concentration and therefore this compound proceeded into early
preclinical/clinical trials [244, 245].
Furthermore, the important interaction between the RSV G-protein and the
cell surface GAG receptor during the virus attachment step have led to the
development of another group of RSV inhibitors consisting of polyanionic
compounds. As discussed in section 1.4 the positively charged GAG binding
domain of the G-protein is important for the interaction with anionic sulfate
groups of the GAG chains. Macromolecular compounds mimicking sulfation
pattern and structure of the GAG chains such as dextran sulfate, heparin,
pentosan sulfate, galactan sulfate and many other polysulfated compounds
have been proven to be efficient inhibitors of infection of cells by RSV and
many other GAG-binding viruses at low microgram/ml concentrations [246-
248, Reviewed in 249]. Although this class of antiviral compounds has the
potential of broad spectrum activity their interactions can be weak and
reversible. Nevertheless, studies show that coupling a lipophilic entity to the
GAG-mimetic could confer a stronger binding and improve antiviral activity
as shown for inhibitors of HIV and HSV [250, 251]. In paper II the coupling
of lipophilic entities to polysulfated tetra- and pentasaccharide glycosides and
its effect on anti-RSV activity was evaluated.
Antiviral drug candidates targeting the post-fusion events in the RSV life
cycle are mainly those interfering with the virus replication including
essential activities of the N- nucleocapsid or the L-polymerase components of
the RSV transcription complex. Compound YM53403 inhibited RSV
infectivity with an IC50 value of 0.2 µM by targeting the viral polymerase
[252]. The 1,4-benzodiazepine candidate drug RSV-604 was shown to target
the N-protein and inhibit RSV replication if added up to six hours after
infection. The compound advanced into phase II clinical trials in 2006 and
was still in phase II in 2010 [253]. In addition to a small-molecule inhibitor
of the N-protein there is also a small interfering RNA (siRNA) based
candidate, ALN-RSV01, which inhibits RSV by interrupting the synthesis of
the N protein. This compound was shown to be active if added up to 24 h p.i.
and is in phase II clinical trials [254, 255].
Candidate antivirals for treatment of respiratory syncytial virus and coronavirus infections
36
Before the emergence of SARS-CoV in 2003, the disease caused by CoVs
was not considered to be severe enough to justify the development of a
vaccine. Following the SARS-CoV outbreak there was an immediate shift in
the efforts to find a vaccine for these severe infections in hopes of preventing
a new outbreak. It has been reported that SARS-CoV induce the production
of protective neutralizing antibodies in infected individuals [256, 257]
indicating that neutralizing antibodies can be used for protective measures. In
contrast, infection with a seasonally circulating CoVs such as 229E does not
provide a strong protective immunity and reinfections are common [258].
Although several vaccine strategies are currently being developed no vaccine
is available for immunization against any CoV.
Several studies indicate that passive administration of neutralizing antibodies
specific for the S-protein can be used as effective immunoprophylaxis or
immunotherapy of CoV infections [Reviewed in 259]. In spite of these efforts
no preparation of CoV-neutralizing antibodies or immune serum is available
for prevention/treatment of CoV infections.
As a consequence of the outbreak of SARS-CoV in 2003, the antiviral
research focused towards this group of viruses has increased immensely.
Since cellular receptors for different CoVs are known to interact with the
viral S-protein [Reviewed in 260] several different antibodies, small-
molecule compounds and peptides were shown to interfere, with varying
efficacy, with the CoV attachment and entry steps [261-263]. Anti-CoV
candidate drugs targeting other structural proteins, i.e. E-, M- and N-proteins
have also been reported with focus on specific siRNAs inhibiting the
expression of these proteins and preventing their important roles in the viral
life cycle [Reviewed in 264].
The major portion of the anti-CoV strategies developed so far have focused
on interference with important CoV enzymatic activities held by the three
virus nsp proteases, i.e., the 3CLpro (nsp5), PL1pro and PL2pro (nsp3), as
well as the 5’-3’ helicase (nsp13) and the RdRp (nsp12) [Reviewed in 264].
The crystal structure of the main CoV protease, 3CLpro [265] revealed
intricate structure details and allowed for a range of modeling techniques to
be used in the search for potential antiviral compounds. The essential
proteolytic activity of the 3CLpro in all CoVs and its conserved structure
imply the possibility of developing a broad spectrum CoV inhibitor [266]. In
Anna Lundin
37
fact several candidate drugs targeting this protease such as the dipeptide
inhibitor JMF1521 and others have been identified [267] and are listed in
Table 1.[268, 269].
Strategies targeting the enzymatic functions of CoV helicase and RdRp have
also been pursued. An example is the inhibition of SARS-CoV helicase by a
group of adamantine-derived bananins with fairly good potency in the
micromolar range [270]. Due to the conserved nature of these proteins their
inhibitors may have a broad CoV-spectrum potential, however, so far no
sufficiently strong antiviral candidate has been identified [271]. Although
CoVs express 16 nsps only a few of them exhibiting enzymatic activities
were targeted by antivirals, some of which are shown in Table 1. In paper
IV, we report for the first time on the identification of an inhibitor of nsp6
activity in 229E-CoV and other human and animal CoVs.
Candidate antivirals for treatment of respiratory syncytial virus and coronavirus infections
38
Table 1. Anti-RSV and anti-CoV candidates
Target class Drug/
Candidate
name
Development
status
Target/mode of action Ref
RSV Prophylactic
monoclonal anti-
F antibody
Palivizumab Approved 1998 Binds to epitopes on the
F1 subunit
[226]
Nucleoside
analogue
Ribavirin Approved 1980
(inhalant form)
Replication inhibitor [272]
Fusion inhibitor TMC-353121 Phase II Binds to the heptad
repeat regions
[239]
Fusion inhibitor BMS-433771 Phase I,
discontinued
Binds to epitopes on the
F1 subunit
[238]
Fusion inhibitor VP-14637 Phase I,
discontinued
HR2 and intermediate
domains of the F1
subunit
[240]
Fusion inhibitor RFI-641 Preclinical,
discontinued
Binds to epitopes on the
F1 subunit
[273]
Attachment
inhibitor
MBX-300
(NMSO3)
Preclinical Conserved regions of
Protein G
[245,
274]
Polymerase (L)
inhibitor
YM-53403 Preclinical Transcription/replication
inhibitor targeting L-
polymerase
[252]
Nucleocapsid (N)
protein inhibitor
ALN-RSV01 Phase II siRNA, prevent the
synthesis of N-protein
[254]
Nucleocapsid (N)
protein inhibitor
RSV-604 Phase II N-protein inhibitor [275]
CoV Protease inhibitor JMF1521 Unknown Competitive inhibitor of
SARS-CoV 3CLpro
[267]
Protease inhibitor Cinanserin Unknown SARS-CoV and 229E
3CLpro inhibitor
[268]
Protease inhibitor Octapeptide
AVLQSGFR
Unknown SARS-CoV 3CLpro
inhibitor
[269]
Nucleoside
analogue
6-azauridine Unknown NL63 replication
inhibitor
[276]
Helicase inhibitor Adamantene-
derived
Bananins
Unknown SARS-CoV helicase
ATPase activity inhibitor
[270]
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High throughput screening (HTS) is a method employed for the screening of
large collections, libraries, of up to over a million compounds where the
effect/activity on a biological process of interest is measured in cluster 96 to
3456 plate formats in a total assay volume of 1-100 µl. This methodology
makes it possible to screen a large number of compounds or factors
simultaneously under the same conditions which also helps to reduce intra-
and inter sample variations making the method robust. In addition, the assays
can be scaled down and hence reduce the amount of material required for the
assays to a minimum. The process can be more or less automated giving
rapid assessment of a large number of factors in a standardized system at a
lower cost [277-282].
Libraries to be used in HTS can contain random small-molecule compounds,
natural product extracts, monoclonal antibodies, siRNAs, biomarkers,
enzymes etc. and can be further divided into categories e.g. based on what
biological activity they target. Selection of small compounds to be included
in the libraries can vary widely but emphasis is put on their “drug-like”
properties and a varying degree of diversity. The compounds with potential
drug-like properties are identified through comparative analysis of medicinal
chemistry databases and drug-like indexes that relates molecular structures of
known drugs with candidate library compounds [283]. The evaluation of the
drug-like potential of a molecule in question is based on analysis of
functional groups and basic chemical properties, often involving the
Lipinski’s rule of five. These set of rules or guidelines help to identify
compounds with desirable qualities in a molecular structure suitable for a
drug [284].
Based on the fact that a number of licensed drugs originate from natural
products, many libraries also contain extracts from plants and microbes with
both land and marine origin, some of which are used in traditional medicine
[285-288]. Due to the complex process of extracting and isolating active
compounds from plants, the focus has previously been on screening of
synthesized molecules. However, with new preparation techniques and the
need for unexplored compound sources more libraries containing natural
products are still emerging [289, 290].
HTS has now developed into one of the most important tools for drug
discovery including the field of antiviral research. In the case of antiviral
Candidate antivirals for treatment of respiratory syncytial virus and coronavirus infections
40
screening, one of the more traditional protocols for whole-virus assays
include testing the effects of library compounds on virus infection of cultured
cells with the use of microscopic analysis to evaluate the protection of cells
from the virus-induced cytopathic effect. With the recent development of
screening systems with more focus on tissue specific primary cells it is also
possible to get a higher physiological relevance in the screens [291]. This
method, although time consuming, allows for differentiation of “true”
antiviral hits from those causing adverse effects on cultured cells. The use of
viral constructs expressing so called reporter proteins such as GFP can
facilitate the reading of HTS assays, however identified hits have to be
reevaluated for their cytotoxicity. Apart from this empiric/random screening,
the HTS can be more rational/specific [292] where the viral target such as
expressed and/or purified viral proteins with enzymatic activity (e.g.
polymerase or neuraminidase) or viral components possessing non-enzymatic
activities (e.g. viral membrane-fusion protein) are screened in cultured cells
or in cell-free system. The results are subsequently acquired with different
read-out techniques, such as spectroscopy/luminescence/fluorescence, FACS
or high content imaging analysis. The basic luminescence/fluorescence read-
outs are commonly used in HTS and are straightforward, inexpensive and
suitable for studying the activity of particular target protein and/or cellular
toxicity. In comparison, HTS read-outs with high content imaging (HCI)
results in large amounts of data from several variables simultaneously that
can be compared across cell populations. The HCI technique has a higher
complexity and efficiency and allows for the analysis of multiple factors and
their correlation. Even so, due to the amount of data generated the analysis is
time consuming and the costs are higher.
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The aim of this work was to develop new antiviral candidates for treatment of
RSV and CoV infections through establishing and using a sequential assay
strategy for their identification and subsequent elucidation of their
mechanism of action. The specific aims were the following:
To identify novel anti-RSV and anti-CoV candidate drugs by
screening large compound collections in a whole virus cell culture-
based system optimized for RSV and CoV.
To establish and optimize an assay-strategy for the step-by-step
evaluation of the mode of antiviral activity of the anti-RSV and anti-
CoV hits.
To assess/elucidate antiviral potency, cytotoxicity and mode of
antiviral activity of the following hits and their analogues.
i. Lipophile-conjugated polysulfated oligosaccharides
as anti-RSV hits
ii. Benzenesulfonamide-based P13 and diazepane-
based C15 anti-RSV hits
iii. Benzamide-based K22 anti-CoV hit
Candidate antivirals for treatment of respiratory syncytial virus and coronavirus infections
42
The compounds screened for anti-RSV and anti-CoV activity in paper I and
IV originated from the ChemBioNet library of 16671 diverse drug-like
compounds obtained from the Leibniz Institute for Molecular Pharmacology
(FMP, Berlin, Germany). Compounds were supplied in a 384-well plate
format at 10 mM concentration in DMSO and were subsequently diluted in
water to appropriate concentrations and stored frozen at -20°C until
screening. Larger quantities of compound K22 were obtained from ChemDiv
(San Diego, California, USA) and subsequently verified for structure and
purity by 1H NMR and LCMS.
The mini-library of polysulfated tetra- and pentasaccharide glycosides,
evaluated in paper II, was obtained from Progen (Australia). Most of these
glycosides were composed of α(1→3)/α(1→2)-linked mannose residues with
a set of varying lipophilic groups attached to the reducing end. These
glycosides and the PG545 glycoside composed of maltotetraose coupled to a
cholestanol group, were all prepared and characterized by 1H NMR,
13C
NMR, mass spectrometric, and microanalytical techniques as described
previously [293, 294]. The precursor compounds for the saccharide fragments
of these glycosides, except for PG545, were the sulfated di- to
hexasaccharides of muparfostat (PI-88). These oligosaccharides were
prepared by sulfonation of phosphomannan, derived from the yeast Pichia
Holstii [295, 296], as described previously [297]. All oligosaccharides and
glycosides were dissolved in water to a final concentration of 10 mg/ml and
stored frozen at -20°C.
Screening of library compounds for anti-RSV activity and other cell-based
assays were conducted using the laboratory strain RSV A2 [298] and human
laryngeal epidermoid carcinoma (HEp-2) or in some assays baby hamster
kidney (BHK-21) cells. They were grown in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 8% fetal calf serum (FCS), 60 µg/ml
of penicillin, 100 µg/ml of streptomycin (PEST), and 8% tryptose–phosphate
broth (for BHK-21).
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Screening for anti-CoV activity and other cell-based assays were conducted
using the laboratory strain of 229E-CoV [25] and human embryonic lung
fibroblasts (MRC-5). The cells were grown in Eagle’s minimum essential
medium (EMEM) supplemented with 10% heat-inactivated fetal calf serum,
(HI-FCS), 1% L-glutamine and PEST. Antiviral testing concerning other
CoVs of human and animal origin were conducted using infectious bronchitis
virus Beaudette (IBV-Beau-R) strain [299] and Vero cells, SARS-CoV strain
Frankfurt-1 [300] and Vero cells, MERS-CoV [33, 301] and well-
differentiated cultures of human airway epithelial (HAE) cells, and
recombinant Renilla luciferase expressing feline CoV (FCoV-Black-Ren)
[302] and feline fetal FCWF-4 cells, Gausia luciferase expressing murine
hepatitis virus (MHV-A59-Gluc) and murine L929 fibroblasts, and Renilla
luciferase-expressing 229E-CoV (229E-CoV-Ren) [303] and HAE cells.
Isolation of normal human bronchial epithelial cells and their subpassaging to
form pseudostratified/differentiated cultures of HAE cells were performed as
described previously [57, 304]. Use of CoV recombinants expressing reporter
luciferases facilitated evaluation of candidate antiviral compounds in HAE
cells.
Screening of the ChemBioNet library for anti-RSV and anti-CoV activity was
conducted in a 384-well plate format in a total volume of 50 µl (Figure 14).
HEp2 or MRC-5 cells were seeded one day prior to the experiment to a
confluency of 60-90%. Appropriate RSV A2 or 229E-CoV concentration for
the assay was determined by the virus stock titration in the 384-well system,
i.e. at the same conditions as the screening assay. Library compounds were
added to cell monolayers at final concentrations of 60 µM and 20 µM for the
anti-RSV and anti-CoV screenings respectively followed by the virus
addition. Cells in some wells received corresponding volumes of DMSO
solvent or water with or without presence of virus to serve as controls. Plates
were incubated at 37°C, 5% CO2, for four days and inspected microscopically
for presence of virus induced cytopathic effect (CPE). Compounds were
evaluated based on their capability of protecting the cells from virus induced
CPE which also provided the first estimation of their specific antiviral
activity and possible adverse effects on cells.
Candidate antivirals for treatment of respiratory syncytial virus and coronavirus infections
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Figure 14. Schematic overview of the compound collection screening. The main screening was
carried out in a 384-well cell culture system seeded with the virus specific susceptible cells.
Primary hits were subsequently assessed in two steps using 24- and 12-well formats,
complemented by evaluation of the hit effect on cell viability and proliferation. Promising hits
were subjected to further assessment of their antiviral mechanism.
Following the primary screening round, candidate hit compounds were
selected for a second screening round in the larger 24-well cell culture format
at a four-level concentration span. Promising hits were subjected to additional
assessment of antiviral activity at fivefold dilutions in 12-well format along
with cytotoxic/cytostatic and proliferation analysis. The antiviral potency of
the compounds were established by determining the concentration inhibiting
the appearance of virus induced CPE by 50%, IC50. More specifically, 100-
200 PFU of RSV or CoV strains and fivefold compound dilutions were added
to cells and incubated for 3h at 37°C. For antiviral activity assessment of the
GAG-mimetics in paper II the compounds were pre-incubated with the virus
for 10 min at room temperature before addition to HEp-2 cells. This step was
followed by removal of the inoculum and an additional 2-3 days of
incubation under a methyl cellulose overlay supplemented with
corresponding concentrations of hit compound. The overlay restricts the virus
infection to the cell-to-cell spreading resulting in formation of viral plaques
which are subsequently visualized by crystal violet staining.
Antiviral potency of hits against other CoVs was assessed in their respective
susceptible cells (see subsection 3:2) using the virus yield reduction assay.
Hit compounds at twofold dilutions were added to susceptible cells at 4 h
prior to the 1 h period of their inoculation with the virus. This step was
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followed by removal of the inoculum, rinsing and addition of assay medium
containing corresponding concentrations of compound. The infectious culture
medium was harvested and the effect of hit compounds on the propagation of
CoVs was monitored either by quantification of amount of viral genomic
RNA (SARS- and MERS-CoV), by measuring luciferase expression (MHV-
A59-Ren, FCoV-Ren and 229E-Ren), or by TCID50 determination in chicken
embryo kidney cells (IBV-CoV).
The potential negative impact of the antiviral candidates on cells was
assessed by tetrazolium-based (MTS) cytotoxicity assay and the proliferation
assays. Toxicity of the tested candidates was expressed as the concentration
of compound reducing cell viability or cell proliferation by 50%, CC50. HEp-
2 or MRC-5 cell monolayers in 96-well plates were supplemented with test
compounds at fivefold concentrations at a range of 0-500 µM or µg/mL for
48-72h at 37°C. The compound cytotoxicity was estimated by the addition of
the MTS reagent for 1-2 h. MTS is metabolized into a colored formazan
product by viable cells and was detected by spectrophotometric measurement
at an absorbance of 490 nm against a background of 650 nm. Experimental
procedure for the cell proliferation assay was similar to the cytotoxicity with
incubation of serially diluted compounds for 48-72h at 37°C. Cells were
subsequently dissociated with EDTA/trypsin and counted. This allows for
assessment of possible cytostatic activity of test compounds. Cells incubated
with corresponding amounts of DMSO solvent or water was included as
controls.
Estimation of compound cytotoxicity and impact on cell viability on Vero,
L929, FCFW and HAE cultures was assessed using the luminescence assay
kits CytoTox-Glo™ and CellTiter-Glo® (Promega) based on the live cell
membrane permeability and the ATP quantification respectively.
Following primary and secondary screening rounds identified anti-RSV and
anti-CoV hits were subjected to elucidation of their mechanism of antiviral
activity (mode-of-action). All methods described in Figure 15 were used as a
step by step elucidation of putative mechanisms of antiviral activity. Each
Candidate antivirals for treatment of respiratory syncytial virus and coronavirus infections
46
assay provided information aiding identification of the antiviral target. For a
more detailed description see papers III and IV.
Firstly, the time-of-addition/removal assay provided initial guidance of
whether the hit compound targets the cell, the viral particle, or the virus
replication in cells (Figure 15). The time of addition/removal assay evaluates
the extent of inhibition of virus infection when the hit compound is added to
MRC5 or HEp-2 cells at different time points relative to the virus
inoculation. In particular, hit compound was added to and incubated with
cells for periods of 2 h occurring either before, during or after a 2 h
inoculation of cells with the virus. Following each period of incubation of
cells with hit compound and/or the virus, the medium was removed, the cells
rinsed and overlaid with methylcellulose, and incubated for 3 days at 37°C.
The viral plaques were visualized by staining with crystal violet. Potent
activity of a hit whose presence on cells was restricted to a short period
before inoculation with the virus strongly suggests that this compound targets
cellular receptor sites for the virus. In contrast, the lack of antiviral activity at
this period and substantial inhibition during co-incubation of the hit-virus
mixture with cells indicate that hit compound targets the viral particle and
inactivate its infectivity with or without affecting activities of the virus
attachment/entry proteins. To discriminate between these possibilities the
activity of hit compound is challenged in virucidal, attachment and fusion
assays. A hit showing the most potent activity when present after the virus
inoculation of cells usually targets the post-entry events of infection.
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Figure 15. Assay strategy for the elucidation of the antiviral mechanism of identified hit
candidates. Initial time of addition/removal assays resulted in an indication of when during the
virus life cycle the compounds had their most potent inhibitory activity and whether it mainly
targeted the virus particle or cell components. Analysis of the compound antiviral mechanism
by this step-by-step assay strategy generated information on specific viral components
involved and their roles in the viral life cycle. The preparation of the drug resistant viral
variants and their subsequent analysis revealed more information on the virus escape
mechanisms and potential alterations in the virus morphology or infection pattern.
Candidate antivirals for treatment of respiratory syncytial virus and coronavirus infections
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Virucidal, or the virus-neutralization, assay helps to clarify whether hit
compounds irreversibly inactivates infectivity of RSV or 229E-CoV
particles. This assay relies on mixing of virus at a high PFU of 105/0.5 mL
with the hit at three different concentrations, 100, 10 and 1 µM or µg/mL, or
with a diluent medium to serve as control. The virus-hit mixture was then
incubated in a 37°C water bath for 15 min followed by the immediate serial
tenfold dilutions of the mixture to a non-inhibitory concentration of hit and
their addition to MRC-5 or HEp-2 cells for plaque titration of the virus.
Decreased viral infectivity as related to the control sample indicates
virucidal/virus-neutralizing activities of the hit. As mentioned previously
apart from disruption of viral particles (virucidal effect) their neutralization
may rely on affecting the activities of viral attachment or fusion proteins in
their native or active intermediate forms.
Compounds identified as potentially targeting the viral particle by the time-
of-addition/removal assay were subjected to elucidation of their specific
impact on viral attachment and fusion activities. These assays were utilized
for the identified anti-RSV hits in paper I and II. All steps of the attachment
assay were carried out in cold room conditions (~4°C). RSV particles to be
used in the assay were metabolically radiolabelled with the 35
S-
methionine/cysteine and then purified by centrifugation on a three-step
discontinuous sucrose gradient. Serial fivefold dilutions of hit compounds in
cold medium were mixed with the radiolabelled virus and incubated for 5
min prior to the addition of the mixture to HEp-2 cells monolayers. After a 2
h inoculation period at 4°C the cells were extensively rinsed, lysed, and
transferred to scintillation vials for quantification of radioactivity. Substantial
decrease of bound viral radioactivity levels compared to the non-treated
control indicates that the hit interferes with the attachment of viral particles to
cells.
The candidate impact on virus mediated fusion was studied through the use
of a firefly luciferase reporter assay. One set of BHK-21 cells was infected
with RSV A2 strain and 24 h later transfected with a plasmid expressing
firefly luciferase under the control of a T7 promoter while another set of
BHK-21 cells were transfected with a plasmid expressing a T7 RNA
polymerase [305, 306]. A principle of this assay is that, the RSV-mediated
fusion of these two sets of cells results in expression of the firefly luciferase
and thus the development of luminescence. Hit compounds at serial fivefold
dilutions were added to the cells infected with RSV and transfected with the
T7 promoter controlled plasmid and incubated for 15 min at 37°C. Cells
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expressing the T7 RNA polymerase were dissociated with EDTA/trypsin and
added to the hit treated cells. This step was followed by co-incubation of cells
for 6 h 37°C, after which the cells were lysed and the levels of luminescence
measured. Decreased level of luminescence as related to the mock-treated
sample indicates that the hit compound is a fusion inhibitor.
Hits that in the time-of-addition/removal assays were identified as post-viral
entry inhibitors were further assessed in the classical time-of-addition assay
to identify the time-span or the latest addition time relative to inoculation of
cells at which the hit still retains antiviral activity. Using this assay the
impact of anti-CoV hit K22 on viral RNA synthesis was studied in paper IV.
More specifically, 229E-CoV was adsorbed to precooled MRC-5 cells for 45
min at 4°C after which the inoculum was removed and cells rinsed. Hit K22
(10 µM) was added at 2 h intervals from 0-12 h relative to the end of the
virus adsorption period and all samples were harvested at 24 h p.i. followed
by the quantification of viral RNA by reverse transcriptase PCR (RT-PCR).
Estimation of the time-span for the most potent hit activity may also provide
a hint for its possible post-entry target that can be assessed by analysis of
viral RNA or protein production, or replication kinetics.
Following assessment of the step of the viral life cycle targeted by hit
compounds additional assays were used for more specific identification of the
antiviral target. Through the preparation and analysis of drug resistant viral
variants specific gene or protein targets could be identified. Resistant virus
variants were obtained through the 10-13 consecutive passages of RSV in
HEp-2 or 229E-CoV in MRC-5 cells in the presence of fixed concentrations
of hits P13 and C15 in paper I or increasing concentrations of hits PG545 in
paper II and K22 in paper IV. Similar passages of the virus in the absence
of hits were also performed to serve as controls for comparative analysis of
the drug resistant viruses. Viral variants that resisted the selective pressure
from the hits were plaque purified followed by estimation of their respective
resistance compared to original and mock passaged virus by the plaque
reduction assay as described in subsection 3.3. Resistant variants were then
subjected to nucleotide sequence analysis through RNA purification of virus
material, cDNA preparation by RT-PCR and subsequent sequencing with
amplification of DNA fragments of the coding regions of the RSV F- and G-
proteins and the 229E-CoV complete genome. Sequences were analyzed by
Candidate antivirals for treatment of respiratory syncytial virus and coronavirus infections
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Sequencher 4.9 software. Presence of a specific aa alteration(s) in at least
several plaque purified drug resistant virus variants suggests the involvement
of the mutated viral component in virus resistance to the hit compound.
A question as to whether the identified aa alteration is indeed responsible for
the drug resistance of the virus needs to be confirmed by preparation of
recombinant viruses harboring this specific aa mutation. This was performed
to confirm resistance of 229E-CoV variants to the K22 hit in paper IV.
Three different variants of the gene fragment comprising resistance specific
mutations were cloned into a TOPO-TA plasmid vector and subsequently
used for recombination into a vaccinia virus system expressing 229E-CoV
[307]. This reverse genetics system produced fully viable recombinant 229E-
CoV with alterations in the specific target protein. Confirmation of resistance
of the recombinant variants in comparison to wild type virus was conducted
by the plaque reduction assay.
Replication fitness of the drug resistant recombinant viruses was assessed in
the replication kinetics and EM assays. Concentrated preparations of wild
type 229E-CoV and recombinant viruses to be used in the replication kinetics
studies were prepared by centrifugation of infectious supernatant medium
through the layer of 20% sucrose. To study the replication fitness, 229E-CoV
was adsorbed to precooled monolayers of MRC-5 cells for 1 h at 4°C,
followed by removal of inoculum, cell rinsing and addition of fresh warm
medium. Infected cells and supernatant fluid were harvested at different time
points of their incubation at 37°C (0-72 h) relative to the end of virus
inoculation. The amount of viral RNA produced in supernatant fluid and in
cells was quantified by RT-PCR. Virus infectivity was quantified in samples
of infectious culture medium by the viral plaque assay.
Electron microscopy (EM) imaging of cells infected with the drug resistant
viral variants may help to analyze replication fitness of these viruses. For this
analysis, MRC-5 cells seeded on melinex polyester film were infected with
wild type or recombinant K22 resistant 229E-CoVs with or without the
presence of K22 hit for 18 h at 37°C. The cells were subsequently fixed with
glutaraldehyde and processed for EM as described by Widehn and Kindblom
[308]. By studying potential changes in the infection pattern of the
recombinant virus compared to the wild type infection, the expression of
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genes with compound specific mutations could be related to phenotypic
alterations in the infected cells.
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In paper I we identified two novel anti-RSV hits targeting the viral
fusion/entry process. These hits were identified by screening of the
ChemBioNet collection of compounds using a 384-well HEp2-cell culture
based screening method, a comprehensive protocol of which is described in
paper III. Preliminary and secondary screening for compound prevention of
the virus induced cytopathic effect resulted in the selection of 221 and
subsequently 13 hits respectively. The two hits that displayed the most
promising inhibitory qualities, namely the N-(2-hydroxyethyl)-4-methoxy-N-
methyl-3-(6-methyl[1,2,4]triazolo[3,4-a]phthalazin-3-yl)benzenesulfonamide
P13 and the 1,4-bis(3-methyl-4-pyridinyl)-1,4-diazepane C15 (Figure 16)
were selected for further assessment of the antiviral potency and cytotoxicity
as well as elucidation of their antiviral mechanisms.
Figure 16. Structure of the diazepane-based C15 (A) and the benzenesulfonamide-based P13
(B) with indications of the different groups of the P13 molecule subjected to structure-activity
assessment by analysis of analogs. Substituents at group 3 that improved the anti-RSV activity
of P13 are depicted (C).
Both compounds exhibited anti-RSV capability in the submicromolar range
with IC50 values, determined by the RSV plaque reduction assay in HEp-2
cells, at concentrations of 0.11 and 0.13 µM for P13 and C15 respectively. In
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combination with cytotoxicity assessment the selective index values were
2818 and 577 for P13 and C15 respectively.
P13, displaying the better inhibitory profile of the two hits, was selected for
additional analysis of its structure activity relationship. Results based on the
screening of fifteen commercially available structurally similar compounds
(analogs) identified the specific structures of the molecule important for its
antiviral activity (Figure 16 B and C). Removal of group 1 resulted in
complete loss of activity. Similar results were observed for group 2 since
changing its position on the benzene ring, replacement by a methyl group or
complete removal resulted in a marked reduction or abolished anti-RSV
activity. Substitution of group 3 demonstrated a way of improving the
compound activity since replacing it with any of the three groups shown in
figure 16 C resulted in a ~2.5 fold improvement in antiviral activity.
An initial elucidation of the antiviral mechanism of the hits aimed at defining
a stage of the RSV life cycle where these compounds were most active. This
was assessed by a time-of-addition/removal assay where P13 and C15 were
added to the cells at different time points relative to the virus inoculation.
Both compounds most potently inhibited RSV infection when present during
the virus inoculation of cells indicating their interference with viral
components during the RSV attachment to and entry into the cells (Figure 17
A-B).
Figure 17. Hits P13 and C15 exhibited
the most pronounced anti-RSV activity
when present during initial RSV infection
of cells as evaluated by the time of
addition/removal assay. P13, C15, or
DMSO solvent was added to HEp-2 cells
at different time points,
before/during/after, relative to the virus
inoculation. Presence of hits on cells was
limited to a 2h period (A) or extended
until the development of viral plaques
(B).
Candidate antivirals for treatment of respiratory syncytial virus and coronavirus infections
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Since P13 and C15 interfered with early events of the RSV life cycle, we
wanted to discriminate whether these hits showed direct inactivating
(virucidal) properties against viral particles or interfered with the virus
attachment to or entry into cells. Co-incubation of these hits with RSV
particles did not affect their infectivity thus excluding the potential virus
inactivating activity of P13 and C15. Furthermore, evaluation of the
compound impact on the RSV capability to attach to cells showed little or no
interference with this step indicating that neither of the compounds targeted
RSV binding to cell surface receptors. These results further supported an
assumption that P13 and C15 affect the stage of RSV life cycle occurring
after attachment of viral particles to cells.
To identify a specific viral component targeted by P13 and C15, the hit
resistant viral variants were generated. After 10 consecutive RSV passages in
HEp-2 cells in the presence of continuous selective pressure generated by 10
µM of respective hit, RSV variants that were at least 1000 times less sensitive
to P13 and C15 than the wild type RSV were selected. Such an extended drop
in hit sensitivity of selected RSV variants suggests a high target specificity of
these hits. Comparative nucleotide sequence analysis of a number of plaque
purified resistant RSV variants, original virus, and a mock passaged virus
revealed mutations in the RSV fusion component, the F-protein. Variants
resistant to P13 displayed mutations in the cysteine-rich (T400I) and the HR1
(N197T) regions while C15-resistant variants all carried mutations at position
D489G in the HR2 domain (Figure 18). The fusion process of RSV is a
critical step in the viral life cycle where the F-protein through a series of
conformational events mediates the fusion of viral and cellular membranes to
deliver the genetic material to the cytoplasm. The critical importance of
fusogenic activity of the F-protein during RSV infection of cells has made it
an attractive target for antiviral intervention [309]. Note that alterations in the
F-protein conferring resistance to P13 and C15 were located within the same
mutation-rich domains found in RSV variants resistant to other fusion
inhibitors. (Figure 18, Table 1). It should be emphasized that these domains
mediate key functions of the F-protein, and most mutations were located in or
near the HR2 region or in the FP. This confirms that these regions are
important for the F-protein fusion activity, and also reveal the most frequent
mutational “escape routes” in the RSV attempt to evade an inhibitor. Since
P13 and C15 did not show direct virucidal activity it is unlikely that these
inhibitors target the native/prefusion form of the F-protein. Instead, an
intermediate form of this protein, where the two HR regions are transiently
exposed to mediate the virus-cell fusion, are more likely to be targeted [103].
C15 and P13 may bind to HR1/HR2 and inhibit viral fusion by preventing the
required affinity interaction between these two regions, hindering the
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apposition of the two membranes, or stabilizing the intermediate in an
extended form of the F-protein which would then be unable to proceed into
the collapsed hairpin structure of the post-fusion form (Figure 13).
Figure 18. Schematic illustration of the disulfide linked subunits F1 and F2 of the RSV F-
protein and the sequential order of the essential regions including the fusion peptide (FP),
heptad repeat 1 (HR1), cysteine-rich region (CR), HR2, transmembrane region (TMR), and
cytoplasmic tail (CT). Indicated are the mutations identified in P13 and C15 resistant RSV
variants, and in RSV variants resistant to fusion inhibitors reported by others (see text above).
To further validate the F-protein as a target of P13 and C15, these hits were
evaluated for their capability to prevent the F-protein mediated fusion by
using a luciferase reporter activation assay in BHK-21 cells. P13 and C15
displayed a dose dependent inhibition of the RSV induced cell-to-cell fusion
activity confirming their role as potent fusion inhibitors (Figure 19).
Together, potent anti-RSV activity and low cytotoxicity of P13 and C15
fusion inhibitors warrant further investigation of these hits as potential drugs
for treatment of RSV disease.
Figure 19. P13 and C15 prevent
the RSV mediated cell-to-cell
fusion of BHK-21 cells. The
luciferase activity, induced by the
RSV mediated fusion of cells, is
expressed as a percentage of the
number of relative light units
(RLU) detected in the presence of
hit relative to the mock-treated
controls.
Candidate antivirals for treatment of respiratory syncytial virus and coronavirus infections
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Antiviral strategies targeting the attachment and entry events early in virus
infectious cycle have the advantage of the generally low abundance of virus
particles which are affected by an inhibitor prior to the attachment to and
entry into host cells thus preventing virus multiplication and reducing the risk
of host specific adverse events.
It has been shown that RSV interaction with GAG chains on host cell
surfaces is important to facilitate the virus binding and entry events which
provides an opportunity to use GAG-mimetics such as sulfated oligo- and
polysaccharides for antiviral intervention. The main mechanism through
which negatively charged GAG mimetics inhibit RSV attachment to cells is
by targeting the positively charged GAG-binding regions of the virus
attachment G-protein, an event that shields the viral protein thus preventing
its interaction with GAG receptors (Figure 20).
Figure 20. Antiviral mechanism of the GAG-mimetics. Indicated in the right panel is a
plausible mechanism behind the improved anti-RSV activity of GAG-mimetics modified with a
lipophilic entity which may be inserted into the viral envelope.
These sulfated polyanionic compounds have been shown to have a broad
spectrum of inhibition among various GAG-binding viruses (HIV, HSV, and
RSV) due to presence of clusters of positively charged aa residues in the
GAG-binding domains of the viral attachment proteins in all these viruses
[248, 310, 311]. The main inhibitory mechanism of GAG-mimetics relies on
electrostatic interactions with the virus attachment proteins. However, these
interactions tend to be weak and reversible [312] resulting in a non-persistent
inhibition that is also sensitive to “dilution effects”. Hence, the compound
needs to be present continuously at high concentrations during the virus
binding stage, and even then it is difficult to reach a complete inhibition of
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the virus infection [313, 314]. Therefore, interactions of GAG mimetics with
the virus attachment proteins need to be altered to achieve a sustainable
inhibition and irreversible inactivation of viral particles.
In paper II we found that sulfated oligosaccharide muparfostat formerly
known as PI-88 exhibited potent anti-RSV activity. Muparfostat was
originally identified as a potent anti-cancer drug [315] but have since then
also been reported to have inhibitory activity against a number of GAG-
binding viruses such as HSV, HIV, dengue and flavivirus [246, 247, 251] as
well as being an antimalarial inhibitor [316]. However the inhibitory effect
on RSV infectivity appeared to be reversible and non-virucidal. Since
previous studies from our laboratory [250] indicated that addition of a
lipophilic group to muparfostat greatly improved its anti-HSV properties, in
paper II we studied the possibility of improving the anti-RSV potency of
muparfostat and other specific sulfated oligosaccharides by their coupling to
various lipophilic structures. Muparfostat, acting as the scaffold for most of
the modifications, consists of a mixture of mainly tetra- and pentasaccharides
which were modified by the conjugation of different lipophilic groups at their
reducing end.
A set of fifteen modified structures were evaluated in a mini-screen at 100
µg/ml for their ability to inhibit RSV infection and compared to unmodified
muparfostat. This initial screen identified four compounds that exhibited
improved antiviral potency manifested as near-complete or complete
inhibition of RSV infectivity and greater virucidal properties (Table 2).
Compounds 14 and PG545, both coupled with cholestanol, exhibited similar
antiviral potency but since PG545 displayed a stronger virucidal activity,
coinciding with the project aim, it was selected for further assessment of
antiviral activity and cytotoxicity in addition to the elucidation of its antiviral
mechanism.
Candidate antivirals for treatment of respiratory syncytial virus and coronavirus infections
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Table 2. Anti-RSV activity of selected oligosaccharide glycosides
Compound
Structure of glycoside Residual
infectivity
(%) b
Virucidal
activityc (%
of residual
infectivity)
Oligo-
saccharide
componenta
(No. of
residues)
Aglycone component
Muparfostat Mainly
penta- and
tetra-
saccharide
None 13.5 ± 5.1 94.0
3 Penta-
saccharide
0.9 ± 1.5 47.3
5 Penta-
saccharide
0.0 57.3
14 Tetra-
saccharide
v
0.0 13.3
PG545 Tetra-
saccharided
0.0 9.9
aManα(1→3)/Manα(1→2)
b Percentage of the number of viral plaques found with drug treated virus (100 µg/ml) relative
to mock treated controls
c Residual RSV infectivity after incubation for 15 min at 37°C of ~105 PFU of the virus with
100 µg/ml of a test compound.
d Composed of α(1→4)-linked glucose residues (maltotetraose).
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Coupling of cholestanol to sulfated tetrasaccharide (PG545) improved the
anti-RSV activity by ~5 times as related to unmodified sulfated
oligosascharide muparfostat. In addition, PG545 reached complete inhibition
of RSV infectivity at 20 µg/ml, a quality that muparfostat did not display
even at 500 µg/ml (Figure 21). Furthermore, while muparfostat completely
lacked virucidal activity at the highest concentration tested, PG545 displayed
strong dose-dependent virus inactivating (virucidal) properties with only
marginal residual infectivity left at 100 µg/ml.
Another interesting feature of PG545 was the potent inhibition of vesicular
stomatitis virus, a Rhabdoviridae family member with documented
sensitivity to GAG-mimetics [317], and lack of inhibitory activity against
influenza A virus that uses sialic acid for its attachment to cells. This further
supported the interpretation that PG545 selectively targets the GAG-binding
viruses.
Since the lipophilic cholestanol group of PG545 may potentially interact with
apolipoproteins in serum, the antiviral and virucidal activity as well as the
Figure 21. Effect of PG545 or
muparfostat on RSV infectivity
and HEp-2 cell viability.
Antiviral and cytotoxicity assays
were conducted with (A) and
without (B) the presence of serum
in culture media. Results are
expressed as a percentage of
mock-treated controls.
Candidate antivirals for treatment of respiratory syncytial virus and coronavirus infections
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cytotoxicity was also evaluated in the absence of serum in the culture
medium. Both the antiviral activity and cytotoxicity was decreased by
approximately five times in the presence of serum. Because of the intended
use of PG545 in the mucus-covered respiratory epithelium, the activity was
also evaluated in the presence of human nasal secretions. This body fluid
decreased anti-RSV activity of PG545, an effect that could be attributed to
the presence of a cholestanol moiety which due to its lipophilic nature is
prone to interaction with other lipophilic structures present in mucus such as
surfactant proteins or free lipids. These unwanted interactions of PG545 that
decreased its anti-RSV activity could be overcome by increasing the initial
PG545 concentration.
Initial evaluation of the PG545 mechanism of anti-RSV action was made
using the time-of-addition/removal assay as previously mentioned for paper I,
and the detailed procedure of this assay is presented in paper III. As
expected PG545 displayed the most potent inhibition when present at the cell
surface at the time of virus inoculation suggesting that this compound mainly
targets early events of RSV infection of cells, but may also interact with
cellular components to a lesser extent.
Furthermore, PG545 prevented the attachment of radiolabeled RSV particles
to cells by ~50%. This partial reduction of RSV binding suggested that the
virucidal activity of this compound could be attributed both to the partial
inhibition of the virus binding to cells and to its interaction with other
components of viral particles such as possible insertion of the cholestanol
moiety of PG545 into viral envelope lipids (Figure 20). However this
interpretation would require further studies.
To identify specific viral components targeted by PG545 and muparfostat, we
attempted to select for the drug resistant RSV variants generated under
selective pressure of these compounds. However, selection for resistant
variants proved to be a challenge and in spite of 13 passages in the presence
of increasing concentrations of PG545 the isolated virus was only 3-4 times
less sensitive to the drug than original virus. In contrast, RSV variants
resistant to muparfostat displayed a 7-9 times reduction in sensitivity after 10
passages. In spite of the relatively low resistance of PG545 selected variants,
nucleotide sequence analysis revealed mutations in the attachment G-protein
in all plaque purified variants examined (Figure 22). These aa alterations
were located in the highly conserved domain in or near the central cysteine
noose of the G-protein suggested to have a role in receptor specificity [82].
Similar analysis for muparfostat resistant variants also revealed a mutation in
the G-protein at aa N191T, slightly downstream of the alterations identified
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in the PG545 variants. This mutation was likely to confer resistance to
muparfostat (Figure 22) and was located in the GAG-binding region of this
protein. The anti-RSV compound NMSO3 (MBX-300) [274] which exhibits
similar structural features to PG545, i.e., it comprises sulfated sialic acid
coupled to two lipophilic acyl chains, targeted the G-protein and the drug
resistant variants comprised a set of mutations in the G-protein including the
F168S aa alteration, which was also detected by us in the PG545 resistant
mutant viruses. Interestingly, coupling of cholesterol to the inhibitory
peptides derived from the fusion proteins of HIV, and Nipah and Hendra
paramyxoviruses has greatly improved their antiviral activities [318, 319].
Figure 22. Schematic representation of the RSV G-protein showing aa alterations identified in
the PG545 and the muparfostat resistant RSV variants.
Together, coupling of a lipophilic entity to polysulfated oligosaccharides
significantly improved their anti-RSV activity. This observation together
with previous reports of this strategy [250, 251] confirms its effectiveness in
improving the inhibitory potential of GAG-mimetics. The cholestanol
component of PG545 not only improved its anti-RSV but also resulted in
emergence of the virus inactivating (virucidal) activity, a feature absent in
muparfostat and other sulfated polysaccharides
Antiviral targeting of the post-entry events of virus life cycle, often
enzymatic activities of viral “replicase” proteins, has proven to be an
efficacious way for treatment of viral diseases.
In the post entry stage of the viral life cycle many viruses establish a site for
their replication inside the cells, usually in the form of an organelle-like
structure. This feature is a classic example of how the viruses in many
Candidate antivirals for treatment of respiratory syncytial virus and coronavirus infections
62
different and remarkable ways can utilize the host resources to their own
advantage. In particular, CoVs recruit intracellular membranes from ER and
modify them to form a complex membrane network with the subsequent
formation of CM, large clusters of DMVs, VPs and other structures described
in section 1.3.3 (Figure 8) [150, 151]. These membrane formations serve as
the virus replication factories providing necessary stability and shelter for this
process.
In paper IV we sought to identify new anti-CoV hit compounds through
screening of the ChemBioNet library using experimental procedures similar
to those described in paper I and III. The 229E-CoV strain was used for this
assay system and the screening strategy resulted in the identification of a
benzamide-based hit, K22, (Figure 23) that in a standard plaque assay in
MRC-5 cells reduced the virus infectivity with IC50 at a concentration of 0.7
µM and a selective index of 157.
The mechanism of action studies, as for those described in paper I and II,
were initiated with a time-of-addition/removal assay. The results of this assay
revealed the most potent activity when K22 was added to cells after their
inoculation with 229E-CoV (Figure 24 A). This suggested that K22 targeted
the post entry stage of CoV cycle, an interpretation further supported by the
lack of the virus-inactivating (virucidal) activity of K22 on viral particles. To
further elucidate the suggested post-entry mode of action, the impact of K22
on viral RNA synthesis was studied. A standard time-of-addition experiment
with addition of compound at 2 h intervals during the first 24 h of 229E-CoV
infection of MRC-5 cells, revealed that the viral RNA and infectivity levels
reached near complete inhibition upon K22 addition up to 6 h p.i. (Figure 24
B, C) thus supporting the early post-entry inhibition mechanism.
Figure 23. Structure of K22, the
(Z)-N-(3-(4-(4-bromophenyl)-4-
hydroxypiperidin-1-yl)-3-oxo-1-
phenylprop-1-en-2-yl)benzamide.
Anna Lundin
63
Figure 24. Elucidation of the K22 mechanism of anti-HCoV-229E activity. The time of
addition/removal assessment indicated that K22 affected post-entry steps (+2) of the HCoV-
229E life cycle (A). Classical time of addition (0-24 h) evaluation showing that K22 most
potently reduced production of viral RNA (B) and infectious virus (C) when added up to 6 h
p.i.
In addition, EM studies were conducted to investigate the effect of K22 on
characteristic structural alterations of MRC-5 cells induced by 229E-CoV. As
shown in Figure 26 A (left panel), the virus induced clusters of DMVs were
clearly seen in the perinuclear area, and clusters of virus particles were found
within ER compartments of MRC-5 cells infected with 229E-CoV in the
absence of K22. In contrast, none of these virus-induced membrane
rearrangements could be observed in cells treated with K22 thus supporting
the idea of inhibition of the CoV life cycle at a post entry step preceding the
assembly of new virions, and likely aimed at the membrane bound replication
process.
For a more precise identification of the K22 target, compound resistant viral
variants were generated during 10-13 consecutive passages of 229E-CoV in
MRC-5 cells under the selective pressure of increasing concentrations of K22
(2-16 µM) in two separate experiments. Subsequent nucleotide sequence
analysis revealed that plaque purified viral variants exhibiting a strong
compound resistance all displayed mutations in the 1ab gene fragment coding
for nsp6. This protein possesses 6-7 hydrophobic TM regions out of which at
least 6 span the lipid bilayer [320]. The H121L and M159V aa changes, each
identified in separate selection experiments, were predicted to be located in
or near the TM regions 4 and 5 of nsp6 (Figure 25).
Candidate antivirals for treatment of respiratory syncytial virus and coronavirus infections
64
Figure 25. Cartoon structure of nsp6 showing location of putative TM regions. Mutations
conferring 229E-CoV resistance to K22, i.e., the H121L and the M159V aa substitutions are
indicated.
CoV nsp6 as well as nsp3 and nsp4 contain multiple hydrophobic domains,
although not all of them are necessarily used as TM regions indicating their
important functions in other hydrophobic interactions [320]. Presence of
multiple TM regions in these proteins strongly suggest their function as an
membrane anchor for other nsps that form the RTC, and their function in
recruitment and modification of cytoplasmic membranes. Recently, it was
suggested that nsp3 and nsp6 have a membrane proliferating function and
that the combined activity of nsp3 and nsp4 could mediate pairing of
membranes [147]. In addition, nsp6 was observed to induce vesicle
formation, a role that has previously been suggested for this protein in
inducing autophagosomes [321].
In addition to 6 TM regions, three luminal loops, and both the N- and C-
terminal tail on the cytosolic side was predicted for nsp6 (Figure 25). The C-
terminal end has been shown to be highly conserved between different CoVs
indicating its important function [322]. A free hydrophobic domain in the C-
terminal tail or an intermediately located hydrophobic region that does not
span the bilayer, could easily interact with other parts of the RTC, connect
apposing membranes into DMV proximity or function in forming the DMVs
[320, 323, 324].
To confirm that the mutations identified in nsp6 confer 229E-CoV resistance
to K22, three recombinant mutant viruses, carrying the nsp6 mutations
individually or combined, were generated by the use of a reverse genetics
vaccinia virus system as described by Thiel et al. [307]. Analysis of
Anna Lundin
65
sensitivity of these recombinants to K22 confirmed the aa alterations H121L
and M159V as being responsible for the drug resistance.
Subsequent studies of the recombinant nsp6 mutant viruses were conducted
to assess whether these specific changes in the nsp6 would affect their
replication kinetics or infectivity. The recombinants showed similar levels of
intra- and extracellular RNA as wild type virus, however the infectivity of the
recombinants was reduced by ~ 34-fold. The results indicate that a native
nsp6 function may not be essential for RNA synthesis but required for
production of a fully infectious virus particle.
Candidate antivirals for treatment of respiratory syncytial virus and coronavirus infections
66
Figure 26. The EM (A) and IF (B) images of MRC-5 cells infected with wild type (WT) 229E-
CoV or recombinant nsp6 mutant viruses H121L, M159V, or H121L/M159V with or without
the presence of K22.
Anna Lundin
67
Additional assessment of the nsp6 recombinants was conducted by EM and
IF imaging analysis. MRC-5 cells infected with wild type 229E or
recombinant viruses with and without the presence of K22 were fixed at 18 h
p.i. and processed for image analysis. For the IF assay CoV nsp8 and dsRNA
were used as markers for the CoV replication complex and viral RNA
synthesis respectively. For wild type 229E-CoV the characteristic staining
pattern of nsp8 and dsRNA was observed only in MRC-5 cells that were not
treated with K22 (Figure 26B) thus confirming a potent and complete
inhibition of CoV-229E replication by K22. Analysis of the three nsp6-
recombinants revealed that these mutant viruses displayed similar IF staining
in both K22 treated and untreated cells thus confirming their K22 resistance,
and also showing that the resistance mutations in nsp6 did not prevent the
formation of the replication transcription complex associated with nsp8.
Furthermore, EM-imaging analysis of the same experimental set-up revealed
a reduced number of virus induced DMVs present in the untreated samples of
the M159V mutant as compared to wild type 229E-CoV (Figure 26A). The
observed reduction of the DMV numbers in the nsp6-recombinant correspond
with the reduction in the mutant specific infectivity and support the idea that
the CoV replication and subsequent virion assembly is firmly connected.
The potential activity of K22 against other CoVs from the alpha, beta and
gamma serogroups was also assessed (Figure 27). K22 displayed inhibitory
activity of varying potency against all CoVs tested with the most potent
inhibition observed for the feline and infectious bronchitis CoVs as well as
for the newly identified human strain MERS-CoV (Figure 27b, d, f). In
addition to the antiviral activity assessment in monolayer cell cultures, the
activity of K22 on 229E-ren, expressing renilla luciferase, and MERS-CoV
strains was also evaluated in differentiated cultures of human airway
epithelium (HAE). These cultures resemble the structure and function of the
native human airways and hence the drug evaluations in these cultures are of
greater biological relevance. Both 229E-CoV-ren and MERS-CoV were
inhibited by K22 in these cultures as assessed by the inhibition of production
of dsRNA. The activity of K22 on MERS-CoV infected HAE cultures was
also visualized by IF (Figure 27g). These results further support that K22 is a
promising candidate drug for the treatment of CoV infections. Furthermore,
assessment of K22 mode of action identified a new druggable target for
CoVs, i.e., a membrane-bound viral RNA synthesis.
Candidate antivirals for treatment of respiratory syncytial virus and coronavirus infections
68
Figure 27. The effect of K22 on diverse animal and human CoVs (a-d). Antiviral activity
(bars) and cell toxicity (data points) of K22 and control (black and white bars respectively)
during murine hepatitis (MHV), feline (FCoV), SARS-CoV and chicken infectious bronchitis
(IBV) CoV infection. Antiviral activity of K22 on HCoV-229E and MERS-CoV in differentiated
human airway epithelial (HAE) cultures (e-f). IF analysis of HAE cultures infected with
MERS-CoV with or without the presence of K22 (g) (TJs stands for the tight junctions
staining)
Anna Lundin
69
In this work screening of collections of compounds using a cell based 384-
well screening system of whole virus led to identification of three novel anti-
RSV and one anti-CoV candidate drugs. By applying a systematic step-by-
step approach with a set of assays suitable for the particular virus studied, the
antiviral mode-of-action of these candidate drugs was elucidated resulting in
(i) identification of novel druggable targets for antiviral intervention and (ii)
extending our knowledge about particular structures or activities essential for
RSV or CoV life cycle.
In particular, P13 and C15 were identified as potent inhibitors of RSV fusion
due to targeting of the two HR domains of the F-protein. These domains are
critical for viral fusion because of their intrinsic capability to forcibly overlap
each other, an event that reduces the length of the virus and cell
interconnecting F-protein thus closely apposing and merging the viral and
cellular membranes. The specific targeting of this essential fusion mechanism
resulted in potent and highly specific inhibition of RSV infectivity in the
submicromolar range without major adverse effects on cells, but
unfortunately also in a rapid generation of the drug resistant RSV variants.
Mutations conferring RSV resistance to P13 and C15, and to fusion inhibitors
reported by others (Table 1), occurred in or near the HR regions of the F-
protein. To circumvent this difficulty we plan to modify the P13 structure to
extend its activity towards more extensive targeting of the HR domain or
according to the multiple target strategy, to supplement P13 treatment with
HR1/HR2-derived peptide analogs that could compete with and block
interaction of native domains.
PG545 glycoside, a conjugate of synthetic sulfated tetrasaccharide and
cholestanol, was found to target the native RSV virions including its
attachment component, the G-protein. Although sulfated oligo- and
polysaccharides are known to be potent inhibitors of the attachment of RSV
to cells and many other GAG-binding viruses, these compounds failed in
large clinical trials to protect women against HIV [203, 325]. One reason
behind this failure is the irreversible and non-virucidal mode of antiviral
activity of these compounds. To circumvent this difficulty we have modified
the structure of these inhibitors by coupling of a lipophilic cholestanol group
to the reducing end of a sulfated oligosaccharide maltotetraose. The resulting
compound, PG545, exhibited enhanced anti-RSV potency including
Candidate antivirals for treatment of respiratory syncytial virus and coronavirus infections
70
emergence of the virus inactivating (virucidal) activity, a feature absent in
native sulfated oligo- and polysaccharides. The use of GAG-mimetics and
their modifications such as PG545 seems to be of special importance in
treatment of RSV disease. These compounds may target the putative receptor
binding region (conserved and cysteine rich) as well as GAG-binding domain
of the G-protein that partly overlaps the “superantigen” sequence believed to
induce an allergy-type Th2 cellular response. PG545 may block these regions
thus preventing the virus attachment to susceptible cells alleviating the Th2
response and thereby the severity of RSV disease. Since RSV infection of the
mucus covered respiratory epithelium is an obvious target for PG545 the
observed tendency of the drug to interact with unspecific lipophilic structures
needs to be considered in future optimizations.
K22 was identified as a potent inhibitor of the membrane-bound viral RNA
synthesis, which represents a new druggable target of the CoV replication
process. Apart from 229E-CoV, the compound also affected infectivity of
potential pandemics-causing SARS- and MERS-CoVs. Little is known about
the specific contributions of the different CoV nsps to the recruitment and
modification of host intracytoplasmic membranes and our results support the
idea of nsp6 being a crucial contributor to this process. Further studies of the
specific interplay between the nsp6 protein and the K22 structure would
reveal more information on the inhibitory activity as well as the function of
nsp6. Notably, even though the double membrane structures characteristic for
CoV infection are not present in cells infected with RSV, this virus also
creates specific replication areas in the cytoplasm, inclusion bodies, which
might be accessible for similar antiviral intervention. K22 is currently
evaluated in an additional structure-activity relationship approach in
combination with optimization of the K22 structure for improved
physiochemical properties. An optimized activity/safety profile will allow for
proceeding with K22 into in vivo studies as a promising candidate for
treatment of CoV infection.
Finally, as already assayed for K22, the in vivo potential of the three anti-
RSV candidates identified in this work should be assessed in a biologically
relevant model of RSV/CoV infection, i.e., in cultures of well-differentiated
airway epithelial cells. These cultures resemble the epithelium of human
airways by comprising fully differentiated ciliated and mucus-producing
secretory cells, and therefore are of obvious importance in validation of
candidate drugs against respiratory viruses.
71
I would like to give my sincerest thanks to everyone that made this thesis
possible
To my main supervisor Edward Trybala, for being a great mentor, pedagogue
and inspiration, and for always being keen on discussions. Thank you for all
the stories!
Tomas Bergström, my co-supervisor, for the endless optimism and
enthusiasm, and for always being encouraging when it comes to new
challenges.
Beata Adamiak, for giving so much of your time and valuable advice. Thank
you for your patience, for good talks and for trying to expand my knowledge
in the Polish language.
Sibylle Widehn, for skillful assistance with the electron microscopy imaging
and many, many hours in the dark.
My co-authors: Nina Kann, Charles Hannoun, Carla Andrighetti-Fröhner,
Loubna Bendrioua, Vito Ferro and Volker Thiel and his group at the
Kantonal Hospital, St.Gallen for your contributions to my work and for
fruitful discussions.
Maria Johansson, Carolina Gustafsson, Anette Roth and Ann-Sofie Tylö. The
lab angels and foundation of the ”Virologen-family” at 3:an. Your generosity
and kindheartedness is beyond words. I owe my greatest gratitude and love to
you and you will always be with me as the brightest memory.
Joanna Said, Rickard Nordén, Katarina Antonsdotter and Mona Brantefjord,
my dear friends/roommates, what a journey! For being able to do this with
you guys I feel truly privileged. Joanna, Dr Said, you impress me every day,
you are a force of nature! Thank you for all the pep-talks, for leading the way
and for the help in “zombie-land”. Rickard, for always being ready to help
and for never missing out on a discussion, no matter what the topic is. Thank
you for all the fun and crazy times! Katarina, you inspire me with your
incredible strength and kindness. Thank you for the great support and
awesome dance moves. Mona, my “partner in craziness”, for being a ray of
sunshine. For the support, great laughs and hockey nights.
Candidate antivirals for treatment of respiratory syncytial virus and coronavirus infections
72
Sebastian Malmström, for always making me smile and for being a portal to
fantastic music, language and culinary experiences. Thank you for all the
great talks, laughs and good times.
My fellow PhD, post-docs and lovely third floor co-workers Kristina
Nyström, Maria Andersson, Simon Larsson, Linn Persson, Esther Nuñez,
Mia Ekblad, Galia Askarieh, James Ayukekbong, Charlotta Eriksson, Nancy
Nenonen, Marie Karlsson, Elin Andersson, Peter Norberg, Ka-Wei Tang,
Johan Aurelius, Jonas Söderholm. For sharing everyday life at the lab and
outside for barbeques, movies, miniature golfing and after works. Nina also
for sharing many early hours in the gym and for inspiring me to try out a lot
of peculiar ways of dividing daily energy.
Sigvard Olofsson, Bo Svennerholm, Jan-Åke Liljeqvist, Staffan Görander,
Magnus Lindh, Johan Westin, Martin Lagging, Helene Norder, Kristoffer
Hellstrand and Peter Horal for contributing to a stimulating atmosphere at the
virology department. Sigvard also for bringing virologists together at the
excellent Smögen Symposiums and for giving us the opportunity to relive old
movie magic.
Zoreh, Annelie, Gerd and Berit, my heroes of the tissue culture department,
for providing me with great material in convenience and inconvenience. For
your flexibility and kindness when I kept you busy with a myriad of cell
lines, thank you!
Dan and Rickard, for being workshop gurus with the tools to fix everything
from incubators to shoes. Dan also for making sure that we get exercise, fish
and are clean before Christmas.
Gaby Helbok, Sabina Wagner and Rita Ehrman our administrative magicians.
Thank you for solving problems and for finding extra time when there is
none. For your patience, kindness and great smiles.
To all other colleagues at the virology department, for creating a great
atmosphere!
All my friends. Trying to express my gratitude to each and every one of you
here in the way you deserve would require an additional book. But I will say
this: You mean everything to me! Thank you for your support, pep-talks,
encouraging mini-movies, baby-bliss and much needed distractions
reminding me that no matter what, life is wonderful with friends like you.
73
My extended families, Lundgren, Glans, Levin, Engström, Leek,
Lundin/Johansson. For all good times and adventures past, present and
future. It has been strengthening to know that you are rooting me on.
My incredible superfamily! For being with me at every step of this journey.
Your unconditional love and support through any endeavor makes me feel
like I can accomplish anything. I love you all so much!
Last but not least, David, for all the love and support! Without you by my
side none of this would have been possible. Shamuushi san!
Candidate antivirals for treatment of respiratory syncytial virus and coronavirus infections
74
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